Methods and devices for treating the skin

ABSTRACT

An apparatus includes multiple first reservoirs and multiple second reservoirs joined with a substrate. Selected ones of the multiple first reservoirs include a reducing agent, and first reservoir surfaces of selected ones of the multiple first reservoirs are proximate to a first substrate surface. Selected ones of the multiple second reservoirs include an oxidizing agent, and second reservoir surfaces of selected ones of the multiple second reservoirs are proximate to the first substrate surface.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. § 119(e) from UnitedStates Provisional Patent Application Ser. Nos. 62/007,295, filed Jun.3, 2014; 62/012,006, filed Jun. 13, 2014; 62/090,011, filed Dec. 10,2014; 62/137,987, filed Mar. 25, 2015; 62/138,041, filed Mar. 25, 2015;and 62/153,163, filed Apr. 27, 2015; the content of each of which isincorporated herein by reference in its entirety.

FIELD

Biologic tissues and cells are affected by a unique electrical stimulus.Accordingly, apparatus and techniques for applying electric stimulus totissue have been developed to address a number of medical issues. Thepresent specification relates to methods and devices useful for treatingskin conditions such as acne.

BACKGROUND

Acne vulgaris is a chronic skin condition characterized by areas ofblackheads, whiteheads, pimples, greasy skin, and possibly scarring. Theresulting appearance may lead to anxiety, reduced self-esteem, anddepression. Genetics is estimated to cause of 80% of cases. The role ofdiet as a cause is unclear, and neither cleanliness nor sunlight appearto be involved. Acne mostly affects skin with a greater number of oilglands; including the face, upper part of the chest, and back. Duringpuberty in both sexes, acne is often brought on by an increase inandrogens such as testosterone. Many treatment options are available toimprove the appearance of acne including lifestyle changes, procedures,and medications.

Acne occurs most commonly during adolescence, affecting an estimated80-90% of teenagers in the Western world. Lower rates are reported insome rural societies. In 2010, acne was estimated to affect 650 millionpeople globally making it the 8th most common disease worldwide. Peoplemay also be affected before and after puberty. Though it becomes lesscommon in adulthood than in adolescence, nearly half of people in theirtwenties and thirties continue to have acne. About 4% continue to havedifficulties into their forties. There are recent reports of emergenceof antibiotic resistant strains of acne-causing bacteria.

Other factors affect the skin as well. For example, aging can causechanges to skin appearance and texture such as wrinkles and sagging.These changes can be related to a number of factors, including theenvironment, a person's genetic makeup or age, and behaviors such as sunexposure or smoking. After age 30, the amounts of collagen, elastin, andhyaluronic acid in and around the skin decrease by about 1-2% per year,and combined with sun damage the loss of these essential components canbe greater than 3% per year. The natural, electrical nature of the skinis slowly diminished over time.

SUMMARY

Aspects disclosed herein include systems, devices, and methods fortreating acne, for example using bioelectric devices that comprise amulti-array matrix of biocompatible microcells and a conductive fluid orcream, for example an anti-acne agent.

Aspects disclosed herein include systems, devices, and methods forrejuvenating skin, for example using bioelectric devices that comprise amulti-array matrix of biocompatible microcells and a conductive fluid orcream.

Aspects disclosed herein comprise bioelectric devices that comprise amulti-array matrix of biocompatible microcells. Such matrices caninclude a first array comprising a pattern of microcells, for exampleformed from a first conductive solution, the solution including a metalspecies; and a second array comprising a pattern of microcells, forexample formed from a second conductive solution, the solution includinga metal species capable of defining at least one voltaic cell forspontaneously generating at least one electrical current with the metalspecies of the first array when said first and second arrays areintroduced to an electrolytic solution and said first and second arraysare not in physical contact with each other. Certain aspects utilize anexternal power source such as AC or DC power or pulsed RF or pulsedcurrent, such as high voltage pulsed current. In one embodiment, theelectrical energy is derived from the dissimilar metals creating abattery at each cell/cell interface, whereas those embodiments with anexternal power source may require conductive electrodes in a spacedapart configuration to predetermine the electric field shape andstrength. The external source could provide energy for a longer periodthan the batteries on the surface.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a detailed plan view of an embodiment disclosed herein.

FIG. 2 is a detailed plan view of a pattern of applied electricalconductors in accordance with an embodiment disclosed herein.

FIG. 3 is an embodiment using the applied pattern of FIG. 2.

FIG. 4 is a cross-section of FIG. 3 through line 3-3.

FIG. 5 is a detailed plan view of an embodiment disclosed herein whichincludes fine lines of conductive metal solution connecting electrodes.

FIG. 6 is a detailed plan view of an embodiment having a line patternand dot pattern.

FIG. 7 is a detailed plan view of an embodiment having two linepatterns.

FIG. 8 depicts embodiments showing the location of discontinuous regionsas well as anchor regions of the system.

FIG. 9 depicts an embodiment showing a mask comprising a multi-arraymatrix of biocompatible microcells and means for securing the mask.

FIG. 10 depicts an embodiment for treating acne of the back.

FIG. 11 depicts an embodiment disclosed herein for localized treatmentof acne. The embodiment includes a hydrated silica pack to maintainhydration of the device.

FIG. 12 depicts PROCELLERA® (an embodiment disclosed herein) output overtime.

FIG. 13 depicts a front view of an embodiment for treating acne of theback.

FIG. 14(A) is an Energy Dispersive X-ray Spectroscopy (EDS) analysis ofAg/Zn BED (“bioelectric device”; refers to an embodiment as disclosedherein).

-   -   a. Scanning Electron Microscope (SEM) image;    -   b. Light Microscope Image;    -   c. Closer view of a golden dot and a grey dot in B respectively.    -   d. Closer view of a golden dot and a grey dot in B respectively.    -   e. EDS element map of zinc;    -   f. EDS element map of silver;    -   g. EDS element map of oxygen;    -   h. EDS element map of carbon. Scale bar A-B, E-H: 1 mm; C-D: 250        μm

14(B,C) Absorbance measurement on treating planktonic PAO1 culture withplacebo, Ag/Zn BED and placebo+Ag dressing; and CFU measurement.

14(D) Zone of inhibition with placebo, Ag/Zn BED and placebo+Agdressing.

FIG. 15 depicts Scanning Electron Microscope (SEM) images of in-vitroPseudomonas aeruginosa PAO1 biofilm treated with placebo, an embodimentdisclosed herein (“BED”), and placebo+Ag dressing. The BED treatedbiofilm shows a dramatic decrease in bacteria number.

FIG. 16 shows extracellular polysaccharide staining (EPS).

FIG. 17 shows live/dead staining. The green fluorescence indicates livePAO1 bacteria while the red fluorescence indicates dead bacteria.

FIG. 18 shows PAO1 staining.

FIG. 19 depicts real-time PCR to assess quorum sensing gene expression.

FIG. 20 shows electron paramagnetic (EPR) spectra using DEPMPO (aphosphorylated derivative of the widely used DMPO spin trap). Spinadduct generation upon exposure to disclosed embodiments for 40 minutesin PBS.

FIG. 21 depicts real-time PCR performed to assess mex gene expressionupon treatment with Ag/Zn BED and 10 mM DTT.

FIG. 22 shows Glycerol-3-Phosphate Dehydrogenase (GPDH) enzyme activity.

-   -   a. OD was measured in the kinetic mode.    -   b. GPDH activity was calculated using the formula,        Glycerol-3-Phosphate dehydrogenase activity=B/(ΔT×V)×Dilution        Factor=nmol/min/ml, where: B=NADH amount from Standard Curve        (nmol). ΔT=reaction time (min). V=sample volume added into the        reaction well (ml).

FIG. 23 shows depicts a skin graft donation site one week afterdonation. The donation site was covered on the left half by an over thecounter solution (TEGADERM®, 3M Company, Saint Paul, Minn.) and on theright half by an LLEC system.

FIG. 24 depicts a subject's back prior to treatment for acne.

FIG. 25 depicts a subject's back after a 2 week application of a devicedisclosed herein; improved acne appearance observed.

FIG. 26 depicts a subject's forehead prior to treatment for acne.

FIG. 27 depicts a subject's forehead after a 2 week application of adevice disclosed herein; improved acne appearance observed.

DETAILED DESCRIPTION

Embodiments disclosed herein include systems that can provide a lowlevel electric field (LLEF) to a tissue or organism (thus a “LLEFsystem”) or, when brought into contact with an electrically conductingmaterial, can provide a low level electric current (LLEC) to a tissue ororganism (thus a “LLEC system”). Thus, in embodiments a LLEC system is aLLEF system that is in contact with an electrically conducting material.In certain embodiments, the electric current or electric field can bemodulated, for example, to alter the duration, size, shape, field depth,duration, current, polarity, or voltage of the system. For example, itcan be desirable to employ an electric field of greater strength ordepth in an area where skin is thicker to achieve optimal treatment. Inembodiments the watt-density of the system can be modulated.

“Acne treatment” refers to any method for reducing the appearance of orpreventing the occurrence of acne. Acne treatment can be applied to anyarea where a subject wishes to reduce or prevent the appearance of acne.Acne treatment methods disclosed herein comprise use of LLEFs and/orLLECs.

“Acne treatment agent” as used herein means substances and devices usedto reduce or prevent acne. They are generally mixtures of chemicalcompounds, some being derived from natural sources, many beingsynthetic. These products are generally liquids or creams or ointmentsintended to be applied to the human body. Examples of acne treatmentagents include, but are not limited to: retinoids such as tretinoin,isotretinoin, motretinide, adapalene, tazarotene, azelaic acid, andretinol; salicylic acid; benzoyl peroxide; resorcinol; sulfur;sulfacetamide; urea; antibiotics such as tetracycline, clindamycin,metronidazole, and erythromycin; anti-inflammatory agents such ascorticosteroids (e.g., hydrocortisone), ibuprofen, naproxen, andhetprofen; imidazoles such as ketoconazole and elubiol; and salts andprodrugs thereof. Other examples of acne treatment agents includeessential oils, alpha-bisabolol, dipotassium glycyrrhizinate, camphor,β-glucan, allantoin, feverfew, flavonoids such as soy isoflavones, sawpalmetto, chelating agents such as EDTA, lipase inhibitors such assilver and copper ions, hydrolyzed vegetable proteins, inorganic ions ofchloride, iodide, fluoride, and their nonionic derivatives chlorine,iodine, fluorine, and synthetic phospholipids and natural phospholipidssuch as ARLASILK™. These products can be electrically conductive. Acnetreatment agents can also include skin treatments such as lasertherapies. Acne treatment agents can include anti-acne agents.

“Activation gel” as used herein means a composition useful formaintaining a moist environment within and about the skin. Activationgels can be conductive.

“Affixing” as used herein can mean contacting a patient or tissue with adevice or system disclosed herein. In embodiments “affixing” can includethe use of straps, elastic, etc.

“Applied” or “apply” as used herein refers to contacting a surface witha conductive material, for example printing, painting, or spraying aconductive ink on a surface. Alternatively, “applying” can meancontacting a patient or tissue or organism with a device or systemdisclosed herein.

“Conductive material” as used herein refers to an object or type ofmaterial which permits the flow of electric charges in one or moredirections. Conductive materials can include solids such as metals orcarbon, or liquids such as conductive metal solutions and conductivegels. Conductive materials can be applied to form at least one matrix.Conductive liquids can dry, cure, or harden after application to form asolid material.

“Cosmetic procedure” as used herein refers to a procedure performed toenhance the appearance of the body. For example, cosmetic proceduresinclude procedures such as dermal filler injection, BOTOX® injection,laser treatments, rhinoplasty, etc.

“Cosmetic product” as used herein means substances used to enhance theappearance of the body. They are generally mixtures of chemicalcompounds, some being derived from natural sources, many beingsynthetic. These products are generally liquids or creams or ointmentsintended to be applied to the human body for cleansing, beautifying,promoting attractiveness, or altering the appearance. These products canbe electrically conductive.

“Discontinuous region” as used herein refers to a “void” in a materialsuch as a hole, slot, or the like. The term can mean any void in thematerial though typically the void is of a regular shape. A void in thematerial can be entirely within the perimeter of a material or it canextend to the perimeter of a material.

“Dots” as used herein refers to discrete deposits of dissimilarreservoirs that can function as at least one battery cell. The term canrefer to a deposit of any suitable size or shape, such as squares,circles, triangles, lines, etc. The term can be used synonymously with,microcells, etc.

“Electrode” refers to similar or dissimilar conductive materials. Inembodiments utilizing an external power source the electrodes cancomprise similar conductive materials. In embodiments that do not use anexternal power source, the electrodes can comprise dissimilar conductivematerials that can define an anode and a cathode.

“Expandable” as used herein refers to the ability to stretch whileretaining structural integrity and not tearing. The term can refer tosolid regions as well as discontinuous or void regions; solid regions aswell as void regions can stretch or expand.

“Galvanic cell” as used herein refers to an electrochemical cell with apositive cell potential, which can allow chemical energy to be convertedinto electrical energy. More particularly, a galvanic cell can include afirst reservoir serving as an anode and a second, dissimilar reservoirserving as a cathode. Each galvanic cell can store chemical potentialenergy. When a conductive material is located proximate to a cell suchthat the material can provide electrical and/or ionic communicationbetween the cell elements the chemical potential energy can be releasedas electrical energy. Accordingly, each set of adjacent, dissimilarreservoirs can function as a single-cell battery, and the distributionof multiple sets of adjacent, dissimilar reservoirs within the apparatuscan function as a field of single-cell batteries, which in the aggregateforms a multiple-cell battery distributed across a surface. Inembodiments utilizing an external power source the galvanic cell cancomprise electrodes connected to an external power source, for example abattery or other power source. In embodiments that areexternally-powered, the electrodes need not comprise dissimilarmaterials, as the external power source can define the anode andcathode. In certain externally powered embodiments, the power sourceneed not be physically connected to the device.

“Matrix” or “matrices” as used herein refer to a pattern or patterns,such as those formed by electrodes on a surface, such as a fabric or afiber, or the like. Matrices can be designed to vary the electric fieldor electric current or microcurrent generated. For example, the strengthand shape of the field or current or microcurrent can be altered, or thematrices can be designed to produce an electric field(s) or current ormicrocurrent of a desired strength or shape.

“Reduction-oxidation reaction” or “redox reaction” as used herein refersto a reaction involving the transfer of one or more electrons from areducing agent to an oxidizing agent. The term “reducing agent” can bedefined in some embodiments as a reactant in a redox reaction, whichdonates electrons to a reduced species. A “reducing agent” is therebyoxidized in the reaction. The term “oxidizing agent” can be defined insome embodiments as a reactant in a redox reaction, which acceptselectrons from the oxidized species. An “oxidizing agent” is therebyreduced in the reaction. In various embodiments a redox reactionproduced between a first and second reservoir provides a current betweenthe dissimilar reservoirs. The redox reactions can occur spontaneouslywhen a conductive material is brought in proximity to first and seconddissimilar reservoirs such that the conductive material provides amedium for electrical communication and/or ionic communication betweenthe first and second dissimilar reservoirs. In other words, in anembodiment electrical currents can be produced between first and seconddissimilar reservoirs without the use of an external battery or otherpower source (e.g., a direct current (DC) such as a battery or analternating current (AC) power source such as a typical electricoutlet). Accordingly, in various embodiments a system is provided whichis “electrically self contained,” and yet the system can be activated toproduce electrical currents. The term “electrically self contained” canbe defined in some embodiments as being capable of producing electricity(e.g., producing currents) without an external battery or power source.The term “activated” can be defined in some embodiments to refer to theproduction of electric current through the application of a radio signalof a given frequency or through ultrasound or through electromagneticinduction. In other embodiments, a system can be provided which includesan external battery or power source. For example, an AC power source canbe of any wave form, such as a sine wave, a triangular wave, or a squarewave. AC power can also be of any frequency such as for example 50 Hz or60 HZ, or the like. AC power can also be of any voltage, such as forexample 120 volts, or 220 volts, or the like. In embodiments an AC powersource can be electronically modified, such as for example having thevoltage reduced, prior to use. In embodiments the electric current canbe pulsed.

“Stretchable” as used herein refers to the ability of embodiments thatstretch without losing their structural integrity. That is, embodimentscan stretch to accommodate irregular skin surfaces or surfaces whereinone portion of the surface can move relative to another portion.

“Skin rejuvenation” as used herein refers to the prevention or reductionof skin irregularities, including rhytides (wrinkles), discoloration(for example, darker areas), roughness in texture, cellulite, and thelike.

LLEC/LLEF Systems and Devices

In embodiments, systems and devices disclosed herein comprise patternedmicro-batteries that create a unique field between each dot pair. Inembodiments, the unique field is very short, i.e. in the range of thephysiologic electric fields. In embodiments, the direction of theelectric field produced by devices disclosed herein is similar tophysiological conditions.

Embodiments disclosed herein can comprise patterns of microcells. Thepatterns can be designed to produce an electric field, an electriccurrent, or both over and through tissue such as human skin. Inembodiments the pattern can be designed to produce a specific size,strength, density, shape, or duration of electric field or electriccurrent. In embodiments reservoir or dot size and separation can bealtered.

In embodiments devices disclosed herein can apply an electric field, anelectric current, or both, wherein the field, current, or both can be ofvarying size, strength, density, shape, or duration in different areasof the embodiment. In embodiments, by micro-sizing the electrodes orreservoirs, the shapes of the electric field, electric current, or bothcan be customized, increasing or decreasing very localized wattdensities and allowing for the design of “smart patterned electrodes”where the amount of electric field over a tissue can be designed orproduced or adjusted based on feedback from the tissue or on analgorithm within sensors operably connected to the embodiment andfed-back to a control module. The electric field, electric current, orboth can be strong in one zone and weaker in another. The electricfield, electric current, or both can change with time and be modulatedbased on treatment goals or feedback from the tissue or patient. Thecontrol module can monitor and adjust the size, strength, density,shape, or duration of electric field or electric current based on tissueparameters. For example, embodiments disclosed herein can produce andmaintain very localized electrical events. For example, embodimentsdisclosed herein can produce specific values for the electric fieldduration, electric field size, electric field shape, field depth,current, polarity, and/or voltage of the device or system.

Devices disclosed herein can generate a localized electric field in apattern determined by the size, distance between, and physicalorientation of the cells or electrodes. Effective depth of the electricfield can be predetermined by the orientation and distance between thecells or electrodes.

Embodiments of the LLEC or LLEF systems disclosed herein can compriseelectrodes or microcells. Each electrode or microcell can be or includea conductive metal. In embodiments, the electrodes or microcells cancomprise any electrically-conductive material, for example, anelectrically conductive hydrogel, metals, electrolytes, superconductors,semiconductors, plasmas, and nonmetallic conductors such as graphite andconductive polymers. Electrically conductive metals can include silver,copper, gold, aluminum, molybdenum, zinc, lithium, tungsten, brass,carbon, nickel, iron, palladium, platinum, tin, bronze, carbon steel,lead, titanium, stainless steel, mercury, Fe/Cr alloys, and the like.The electrode can be coated or plated with a different metal such asaluminum, gold, platinum or silver.

In certain embodiments, dot, reservoir, or electrode geometry cancomprise circles, polygons, lines, zigzags, ovals, stars, or anysuitable variety of shapes. This provides the ability todesign/customize surface electric field shapes as well as depth ofpenetration. For example. In embodiments it can be desirable to employan electric field of greater strength or depth in an area where skin isthicker, for example to achieve optimal acne treatment.

Reservoir or dot sizes and concentrations can be of various sizes, asthese variations can allow for changes in the properties of the electricfield created by embodiments of the invention. Certain embodimentsprovide an electric field at about 1 Volt and then, under normal tissueloads with resistance of 100 k to 300K ohms, produce a current in therange of 1 to 10 microamperes. The electric field strength can bedetermined by calculating ½ the separation distance and applying it inthe z-axis over the midpoint between the cell. This indicates thetheoretical location of the highest strength field line.

A system disclosed herein and placed over tissue such as skin can moverelative to the tissue. Reducing the amount of motion between tissue anddevice can be advantageous to skin treatment. Slotting or placing cutsinto the device can result in less friction or tension on the skin. Inembodiments, use of an elastic dressing similar to the elasticity of theskin is disclosed.

In embodiments the system comprises a component such as an adhesive orstraps to maintain or help maintain its position. The adhesive componentcan be covered with a protective layer that is removed to expose theadhesive at the time of use. In embodiments the adhesive can comprise,for example, sealants, such as hypoallergenic sealants, gecko sealants,mussel sealants, waterproof sealants such as epoxies, and the like.Straps can include velcro or similar materials to aid in maintaining theposition of the device.

In embodiments the positioning component can comprise an elastic filmwith an elasticity, for example, similar to that of skin, or greaterthan that of skin, or less than that of skin. In embodiments, the LLECor LLEF system can comprise a laminate where layers of the laminate canbe of varying elasticities. For example, an outer layer may be highlyelastic and an inner layer in-elastic or less elastic. The in-elasticlayer can be made to stretch by placing stress relieving discontinuousregions or slits through the thickness of the material so there is amechanical displacement rather than stress that would break the fabricweave before stretching would occur. In embodiments the slits can extendcompletely through a layer or the system or can be placed whereexpansion is required. In embodiments of the system the slits do notextend all the way through the system or a portion of the system such asthe substrate. In embodiments the discontinuous regions can pass halfwaythrough the long axis of the substrate.

In embodiments the device can be shaped to fit an area of desired use,for example the human face, or around a subject's eyes, or around asubject's forehead, a subject's cheeks, a subject's chin, a subject'sback, a subject's chest, or any area where acne treatment is desired.For example, in embodiments the device can be shaped to fit an areawhere a subject has visible signs of acne, or where a subject wishes toprevent or reduce the appearance or occurrence of acne. In embodimentsthe device can be shaped to fit an area where a subject has visiblesigns of aging, or where a subject wishes to prevent or reduce theappearance or occurrence of wrinkles.

In embodiments the electric field can be extended, for example throughthe use of a hydrogel. In certain embodiments, for example treatmentmethods, it can be preferable to utilize AC or DC current. For example,embodiments disclosed herein can employ phased array, pulsed, squarewave, sinusoidal, or other wave forms, or the like. Certain embodimentsutilize a controller to produce and control power production and/ordistribution to the device.

Embodiments disclosed herein comprise biocompatible electrodes orreservoirs or dots on a surface or substrate, for example a fabric, afiber, or the like. In embodiments the surface or substrate can bepliable, for example to better follow the contours of an area to betreated, such as the face or back. In embodiments the surface cancomprise a gauze or mesh or plastic. Suitable types of pliable surfacesfor use in embodiments disclosed herein can be absorbent ornon-absorbent textiles, low-adhesives, vapor permeable films,hydrocolloids, hydrogels, alginates, foams, foam-based materials,cellulose-based materials including Kettenbach fibers, hollow tubes,fibrous materials, such as those impregnated with anhydrous/hygroscopicmaterials, beads and the like, or any suitable material as known in theart. In embodiments the pliable material can form, for example, a mask,such as that worn on the face, an eye patch, a shirt or a portionthereof, for example an elastic or compression shirt, or a portionthereof, a wrapping, towel, cloth, fabric, or the like. Embodiments cancomprise multiple layers. Multi layer embodiments can include, forexample, a skin-contacting layer, a hydration layer, and a hydrationcontainment layer.

Embodiments can include coatings on the surface, such as, for example,over or between the electrodes or cells. Such coatings can include, forexample, silicone, and electrolytic mixture, hypoallergenic agents,drugs, biologics, stem cells, skin substitutes, cosmetic products, orthe like. Drugs suitable for use with embodiments of the inventioninclude analgesics, antibiotics, anti-inflammatories, anti-acnemedications, or the like.

In embodiments the material can include a port to access the interior ofthe material, for example to add fluid, gel, cosmetic products, ahydrating material, an anti-acne agent, or some other material to thedressing. Certain embodiments can comprise a “blister” top that canenclose a material such as, for example, an acne treatment agent, a skinrejuvenation agent, or the like. In embodiments the blister top cancontain a material that is released into or on to the material when theblister is pressed, for example a liquid or cream. For example,embodiments disclosed herein can comprise a blister top containing askin treatment product, such as an anti-acne agent or medication, or ahydrating material, or the like.

In embodiments the system comprises a component such as elastic tomaintain or help maintain its position. In embodiments the systemcomprises components such as straps to maintain or help maintain itsposition. In certain embodiments the system or device comprises a strapon either end of the long axis, or a strap linking on end of the longaxis to the other. In embodiments that straps can comprise velcro or asimilar fastening system. In embodiments the straps can comprise elasticmaterials. In further embodiments the strap can comprise a conductivematerial, for example a wire to electrically link the device with othercomponents, such as monitoring equipment or a power source. Inembodiments the device can be wirelessly linked to monitoring or datacollection equipment, for example linked via Bluetooth to a cell phonethat collects data from the device. In certain embodiments the devicecan comprise data collection means, such as temperature, pressure, orconductivity data collection means.

A LLEC or LLEF system disclosed herein can comprise “anchor” regions or“arms” or straps to affix the system securely. The anchor regions orarms can anchor the LLEC or LLEF system. For example, a LLEC or LLEFsystem can be secured to an area proximal to a joint or irregular skinsurface, and anchor regions of the system can extend to areas of minimalstress or movement to securely affix the system. Further, the LLECsystem can reduce stress on an area, for example by “countering” thephysical stress caused by movement.

In embodiments the LLEC or LLEF system can comprise additional materialsto aid in acne treatment or skin rejuvenation. These additionalmaterials can comprise, for example, acne treatment agents, skinrejuvenation agents, or both, or the like. These additional materialscan comprise cosmetic formulations, for example Estee Lauder AdvancedNight Repair, or StriVectin Tightening Neck Cream, or the like.

Embodiments disclosed herein can comprise a cosmetic product. Forexample, embodiments can comprise a skin care cream wherein the skincare cream is located between the skin and the electrode surface.Embodiments disclosed herein can comprise a cosmetic procedure. Forexample, embodiments can be employed before, after, or during a cosmeticprocedure, such as before, after, or during a dermal filler injection.Certain embodiments can comprise use of a device disclosed hereinbefore, after, or during a BOTOX® injection. Certain embodiments cancomprise use of a device disclosed herein before, after, or during adermabrasion procedure. Certain embodiments can comprise use of a devicedisclosed herein before, after, or during a laser resurfacing. Certainembodiments can comprise use of a device disclosed herein before, after,or during a resurfacing procedure.

In embodiments, methods and devices disclosed herein can be used toreduce the visibility of skin facial wrinkles, reduce atrophy, orincrease collagen stimulation. The devices can be used either alone orin conjunction with other components well known in the art, such assubcutaneous fillers, implants, intramuscular injections, andsubcutaneous injections, such as dermal fillers or BOTOX® injection. Forexample, the devices can be used in conjunction with collagen and/orhyaluronic acid injections.

In embodiments, methods for rejuvenating skin comprise the step oftopically administering a skin care material on the skin surface or uponthe matrix of biocompatible microcells. These skin care materials cancomprise, for example, anti-aging formulations such as, for example,Estee Lauder Night Repair; Philosophy Miracle Worker; CliniqueRepairware; Lancome Genifique, Renergie, or Bienfait; Elizabeth ArdenPrevage; Strivectin TL; Clarins Double Serum; Peter-Thomas RothUn-Wrinkle; and the like. In embodiments, the skin care material can bean electrically conductive material.

Embodiments can include devices in the form of a gel, such as, forexample, a one- or two-component gel that is mixed on use. Embodimentscan include devices in the form of a spray, for example, a one- ortwo-component spray that is mixed on use.

In embodiments, the LLEC or LLEF system can comprise instructions ordirections on how to place the system to maximize its performance.

Embodiments can comprise a kit comprising a device disclosed herein anda cosmetic or skin treatment agent. Kits disclosed herein can includedirections for use.

LLEC/LLEF Systems and Devices; Methods of Manufacture

In certain embodiments dissimilar metals can be used to create anelectric field with a desired voltage. In certain embodiments thepattern of reservoirs can control the watt density and shape of theelectric field.

In embodiments printing devices can be used to produce LLEC or LLEFsystems disclosed herein. For example, inkjet or “3D” printers can beused to produce embodiments. In embodiments “ink” or “paint” cancomprise any conductive solution suitable for forming an electrode on asurface, such as a conductive metal solution. In embodiments “printing”or “painted” can comprise any method of applying a conductive materialsuch as a conductive liquid material to a material upon which a matrixis desired, such as a fabric.

In certain embodiments the binders or inks used to produce LLEC or LLEFsystems disclosed herein can include, for example, poly cellulose inks,poly acrylic inks, poly urethane inks, silicone inks, and the like. Inembodiments the type of ink used can determine the release rate ofelectrons from the reservoirs. In embodiments various materials can beadded to the ink or binder such as, for example, conductive or resistivematerials can be added to alter the shape or strength of the electricfield. Other materials, such as silicon, can be added to enhance scarreduction. Such materials can also be added to the spaces betweenreservoirs.

In embodiments, electroceutical fabric embodiments disclosed herein canbe woven of at least two types of fibers; fibers comprising sectionstreated or coated with a substance capable of forming a positiveelectrode; and fibers comprising sections treated or coated with asubstance capable of forming a negative electrode. The fabric canfurther comprise fibers that do not form an electrode. Long lengths offibers can be woven together to form fabrics. For example, the fiberscan be woven together to form a regular pattern of positive and negativeelectrodes.

Certain embodiments can utilize a power source to create the electriccurrent, such as a battery or a microbattery. The power source can beany energy source capable of generating a current in the LLEC system andcan include, for example, AC power, DC power, radio frequencies (RF)such as pulsed RF, induction, ultrasound, and the like.

Dissimilar metals used to make a LLEC or LLEF system disclosed hereincan be silver and zinc, and the electrolytic solution can include sodiumchloride in water. In certain embodiments the electrodes are appliedonto a non-conductive surface to create a pattern, most preferably anarray or multi-array of voltaic cells that do not spontaneously reactuntil they contact an electrolytic solution. Sections of thisdescription use the terms “printing” with “ink,” but it is understoodthat the patterns may instead be “painted” with “paints.” The use of anysuitable means for applying a conductive material is contemplated. Inembodiments “ink” or “paint” can comprise any solution suitable forforming an electrode on a surface such as a conductive materialincluding a conductive metal solution. In embodiments “printing” or“painted” can comprise any method of applying a solution to a materialupon which a matrix is desired.

A preferred material to use in combination with silver to create thevoltaic cells or reservoirs of disclosed embodiments is zinc. Zinc hasbeen well-described for its uses in prevention of infection in suchtopical antibacterial agents as Bacitracin zinc, a zinc salt ofBacitracin. Zinc is a divalent cation with antibacterial properties ofits own.

Turning to the figures, in FIG. 1, the dissimilar first electrode 6 andsecond electrode 10 are applied onto a desired primary surface 2 of anarticle 4. In one embodiment a primary surface is a surface of a LLEC orLLEF system that comes into direct contact with an area to be treatedsuch as a skin surface.

In various embodiments the difference of the standard potentials of theelectrodes or dots or reservoirs can be in a range from about 0.05 V toapproximately about 5.0 V. For example, the standard potential can beabout 0.05 V, about 0.06 V, about 0.07 V, about 0.08 V, about 0.09 V,about 0.1 V, about 0.2 V, about 0.3 V, about 0.4 V, about 0.5 V, about0.6 V, about 0.7 V, about 0.8 V, about 0.9 V, about 1.0 V, about 1.1 V,about 1.2 V, about 1.3 V, about 1.4 V, about 1.5 V, about 1.6 V, about1.7 V, about 1.8 V, about 1.9 V, about 2.0 V, about 2.1 V, about 2.2 V,about 2.3 V, about 2.4 V, about 2.5 V, about 2.6 V, about 2.7 V, 2.8 V,about 2.9 V, about 3.0 V, about 3.1 V, about 3.2 V, about 3.3 V, about3.4 V, about 3.5 V, about 3.6 V, about 3.7 V, about 3.8 V, about 3.9 V,about 4.0 V, about 4.1 V, about 4.2 V, about 4.3 V, 4.4 V, 4.5 V, about4.6 V, about 4.7 V, 4.8 V, about 4.9 V, about 5.0 V, about 5.1 V, about5.2 V, about 5.3 V, about 5.4 V, about 5.5 V, about 5.6 V, about 5.7 V,about 5.8 V, about 5.9 V, about 6.0 V, or the like.

In embodiments, LLEC systems disclosed herein can produce a low levelelectric current of between for example about 1 and about 200micro-amperes, between about 10 and about 190 micro-amperes, betweenabout 20 and about 180 micro-amperes, between about 30 and about 170micro-amperes, between about 40 and about 160 micro-amperes, betweenabout 50 and about 150 micro-amperes, between about 60 and about 140micro-amperes, between about 70 and about 130 micro-amperes, betweenabout 80 and about 120 micro-amperes, between about 90 and about 100micro-amperes, or the like.

In an embodiment, a LLEC system disclosed herein can produce a low levelelectric current of between for example about 1 and about 10micro-amperes

In embodiments, LLEC systems disclosed herein can produce a low levelmicrocurrent of between for example about 1 and about 400 micro-amperes,between about 20 and about 380 micro-amperes, between about 400 andabout 360 micro-amperes, between about 60 and about 340 micro-amperes,between about 80 and about 320 micro-amperes, between about 100 andabout 3000 micro-amperes, between about 120 and about 280 micro-amperes,between about 140 and about 260 micro-amperes, between about 160 andabout 240 micro-amperes, between about 180 and about 220 micro-amperes,or the like.

In embodiments, LLEC systems disclosed herein can produce a low levelmicrocurrent about 10 micro-amperes, about 20 micro-amperes, about 30micro-amperes, about 40 micro-amperes, about 50 micro-amperes, about 60micro-amperes, about 70 micro-amperes, about 80 micro-amperes, about 90micro-amperes, about 100 micro-amperes, about 110 micro-amperes, about120 micro-amperes, about 130 micro-amperes, about 140 micro-amperes,about 150 micro-amperes, about 160 micro-amperes, about 170micro-amperes, about 180 micro-amperes, about 190 micro-amperes, about200 micro-amperes, about 210 micro-amperes, about 220 micro-amperes,about 240 micro-amperes, about 260 micro-amperes, about 280micro-amperes, about 300 micro-amperes, about 320 micro-amperes, about340 micro-amperes, about 360 micro-amperes, about 380 micro-amperes,about 400 micro-amperes, or the like.

In embodiments, the disclosed LLEC systems can produce a low levelmicro-current of not more than 10 micro-amperes, or not more than about20 micro-amperes, not more than about 30 micro-amperes, not more thanabout 40 micro-amperes, not more than about 50 micro-amperes, not morethan about 60 micro-amperes, not more than about 70 micro-amperes, notmore than about 80 micro-amperes, not more than about 90 micro-amperes,not more than about 100 micro-amperes, not more than about 110micro-amperes, not more than about 120 micro-amperes, not more thanabout 130 micro-amperes, not more than about 140 micro-amperes, not morethan about 150 micro-amperes, not more than about 160 micro-amperes, notmore than about 170 micro-amperes, not more than about 180micro-amperes, not more than about 190 micro-amperes, not more thanabout 200 micro-amperes, not more than about 210 micro-amperes, not morethan about 220 micro-amperes, not more than about 230 micro-amperes, notmore than about 240 micro-amperes, not more than about 250micro-amperes, not more than about 260 micro-amperes, not more thanabout 270 micro-amperes, not more than about 280 micro-amperes, not morethan about 290 micro-amperes, not more than about 300 micro-amperes, notmore than about 310 micro-amperes, not more than about 320micro-amperes, not more than about 340 micro-amperes, not more thanabout 360 micro-amperes, not more than about 380 micro-amperes, not morethan about 400 micro-amperes, not more than about 420 micro-amperes, notmore than about 440 micro-amperes, not more than about 460micro-amperes, not more than about 480 micro-amperes, or the like.

In embodiments, LLEC systems disclosed herein can produce a low levelmicrocurrent of not less than 10 micro-amperes, not less than 20micro-amperes, not less than 30 micro-amperes, not less than 40micro-amperes, not less than 50 micro-amperes, not less than 60micro-amperes, not less than 70 micro-amperes, not less than 80micro-amperes, not less than 90 micro-amperes, not less than 100micro-amperes, not less than 110 micro-amperes, not less than 120micro-amperes, not less than 130 micro-amperes, not less than 140micro-amperes, not less than 150 micro-amperes, not less than 160micro-amperes, not less than 170 micro-amperes, not less than 180micro-amperes, not less than 190 micro-amperes, not less than 200micro-amperes, not less than 210 micro-amperes, not less than 220micro-amperes, not less than 230 micro-amperes, not less than 240micro-amperes, not less than 250 micro-amperes, not less than 260micro-amperes, not less than 270 micro-amperes, not less than 280micro-amperes, not less than 290 micro-amperes, not less than 300micro-amperes, not less than 310 micro-amperes, not less than 320micro-amperes, not less than 330 micro-amperes, not less than 340micro-amperes, not less than 350 micro-amperes, not less than 360micro-amperes, not less than 370 micro-amperes, not less than 380micro-amperes, not less than 390 micro-amperes, not less than 400micro-amperes, or the like.

The applied electrodes or reservoirs or dots can adhere or bond to theprimary surface 2 because a biocompatible binder is mixed, inembodiments into separate mixtures, with each of the dissimilar metalsthat will create the pattern of voltaic cells, in embodiments. Most inksare simply a carrier, and a binder mixed with pigment. Similarly,conductive metal solutions can be a binder mixed with a conductiveelement. The resulting conductive metal solutions can be used with anapplication method such as screen printing to apply the electrodes tothe primary surface in predetermined patterns. Once the conductive metalsolutions dry and/or cure, the patterns of spaced electrodes cansubstantially maintain their relative position, even on a flexiblematerial such as that used for a LLEC or LLEF system. To make a limitednumber of the systems of an embodiment disclosed herein, the conductivemetal solutions can be hand applied onto a common adhesive bandage sothat there is an array of alternating electrodes that are spaced about amillimeter apart on the primary surface of the bandage. The solution canbe allowed to dry before being applied to a surface so that theconductive materials do not mix, which could interrupt the array andcause direct reactions that will release the elements.

In certain embodiments that utilize a poly-cellulose binder, the binderitself can have an beneficial effect such as reducing the localconcentration of matrix metallo-proteases through an iontophoreticprocess that drives the cellulose into the surrounding tissue. Thisprocess can be used to electronically drive other components such asdrugs into the surrounding tissue.

The binder can include any biocompatible liquid material that can bemixed with a conductive element (preferably metallic crystals of silveror zinc) to create a conductive solution which can be applied as a thincoating to a surface. One suitable binder is a solvent reduciblepolymer, such as the polyacrylic non-toxic silk-screen ink manufacturedby COLORCON® Inc., a division of Berwind Pharmaceutical Services, Inc.(see COLORCON® NO-TOX® product line, part number NT28). In an embodimentthe binder is mixed with high purity (at least 99.999%) metallic silvercrystals to make the silver conductive solution. The silver crystals,which can be made by grinding silver into a powder, are preferablysmaller than 100 microns in size or about as fine as flour. In anembodiment, the size of the crystals is about 325 mesh, which istypically about 40 microns in size or a little smaller. The binder isseparately mixed with high purity (at least 99.99%, in an embodiment)metallic zinc powder which has also preferably been sifted throughstandard 325 mesh screen, to make the zinc conductive solution. Forbetter quality control and more consistent results, most of the crystalsused should be larger than 325 mesh and smaller than 200 mesh. Forexample the crystals used should be between 200 mesh and 325 mesh, orbetween 210 mesh and 310 mesh, between 220 mesh and 300 mesh, between230 mesh and 290 mesh, between 240 mesh and 280 mesh, between 250 meshand 270 mesh, between 255 mesh and 265 mesh, or the like.

Other powders of metal can be used to make other conductive metalsolutions in the same way as described in other embodiments.

The size of the metal crystals, the availability of the surface to theconductive fluid and the ratio of metal to binder affects the releaserate of the metal from the mixture. When COLORCON® polyacrylic ink isused as the binder, about 10 to 40 percent of the mixture should bemetal for a longer term bandage (for example, one that stays on forabout 10 days). For example, for a longer term LLEC or LLEF system thepercent of the mixture that should be metal can be 8 percent, or 10percent, 12 percent, 14 percent, 16 percent, 18 percent, 20 percent, 22percent, 24 percent, 26 percent, 28 percent, 30 percent, 32 percent, 34percent, 36 percent, 38 percent, 40 percent, 42 percent, 44 percent, 46percent, 48 percent, 50 percent, or the like.

If the same binder is used, but the percentage of the mixture that ismetal is increased to 60 percent or higher, then the release rate willbe much faster and a typical system will only be effective for a fewdays. For example, for a shorter term device, the percent of the mixturethat should be metal can be 40 percent, or 42 percent, 44 percent, 46percent, 48 percent, 50 percent, 52 percent, 54 percent, 56 percent, 58percent, 60 percent, 62 percent, 64 percent, 66 percent, 68 percent, 70percent, 72 percent, 74 percent, 76 percent, 78 percent, 80 percent, 82percent, 84 percent, 86 percent, 88 percent, 90 percent, or the like.

For LLEC or LLEF systems comprising a pliable substrate it can bedesirable to decrease the percentage of metal down to, for example, 5percent or less, or to use a binder that causes the crystals to be moredeeply embedded, so that the primary surface will be antimicrobial for avery long period of time and will not wear prematurely. Other binderscan dissolve or otherwise break down faster or slower than a polyacrylicink, so adjustments can be made to achieve the desired rate ofspontaneous reactions from the voltaic cells.

To maximize the number of voltaic cells, in various embodiments, apattern of alternating silver masses or electrodes or reservoirs andzinc masses or electrodes or reservoirs can create an array ofelectrical currents across the primary surface. A basic pattern, shownin FIG. 1, has each mass of silver equally spaced from four masses ofzinc, and has each mass of zinc equally spaced from four masses ofsilver, according to an embodiment. The first electrode 6 is separatedfrom the second electrode 10 by a spacing 8. The designs of firstelectrode 6 and second electrode 10 are simply round dots, and in anembodiment, are repeated. Numerous repetitions 12 of the designs resultin a pattern. For an exemplary device comprising silver and zinc, eachsilver design preferably has about twice as much mass as each zincdesign, in an embodiment. For the pattern in FIG. 1, the silver designsare most preferably about a millimeter from each of the closest fourzinc designs, and vice-versa. The resulting pattern of dissimilar metalmasses defines an array of voltaic cells when introduced to anelectrolytic solution. Further disclosure relating to methods ofproducing micro-arrays can be found in U.S. Pat. No. 7,813,806 entitledCURRENT PRODUCING SURFACE FOR TREATING BIOLOGIC TISSUE issued Oct. 12,2010, which is incorporated by reference in its entirety.

A dot pattern of masses like the alternating round dots of FIG. 1 can bepreferred when applying conductive material onto a flexible material,such as those used for a facial or eye mask, or an article of clothingsuch as a shirt or shorts, as the dots won't significantly affect theflexibility of the material. To maximize the density of electricalcurrent over a primary surface the pattern of FIG. 2 can be used. Thefirst electrode 6 in FIG. 2 is a large hexagonally shaped dot, and thesecond electrode 10 is a pair of smaller hexagonally shaped dots thatare spaced from each other. The spacing 8 that is between the firstelectrode 6 and the second electrode 10 maintains a relativelyconsistent distance between adjacent sides of the designs. Numerousrepetitions 12 of the designs result in a pattern 14 that can bedescribed as at least one of the first design being surrounded by sixhexagonally shaped dots of the second design.

FIGS. 3 and 4 show how the pattern of FIG. 2 can be used to make anembodiment disclosed herein. The pattern shown in detail in FIG. 2 isapplied to the primary surface 2 of an embodiment. The back 20 of theprinted material is fixed to a substrate layer 22. This layer isadhesively fixed to a pliable layer 16.

FIG. 5 shows an additional feature, which can be added between designs,that can initiate the flow of current in a poor electrolytic solution. Afine line 24 is printed using one of the conductive metal solutionsalong a current path of each voltaic cell. The fine line will initiallyhave a direct reaction but will be depleted until the distance betweenthe electrodes increases to where maximum voltage is realized. Theinitial current produced is intended to help control edema so that theLLEC system will be effective. If the electrolytic solution is highlyconductive when the system is initially applied the fine line can bequickly depleted and the device will function as though the fine linehad never existed.

FIGS. 6 and 7 show alternative patterns that use at least one linedesign. The first electrode 6 of FIG. 6 is a round dot similar to thefirst design used in FIG. 1. The second electrode 10 of FIG. 6 is aline. When the designs are repeated, they define a pattern of parallellines that are separated by numerous spaced dots. FIG. 7 uses only linedesigns. The first electrode 6 can be thicker or wider than the secondelectrode 10 if the oxidation-reduction reaction requires more metalfrom the first conductive element (mixed into the first design'sconductive metal solution) than the second conductive element (mixedinto the second design's conductive metal solution). The lines can bedashed. Another pattern can be silver grid lines that have zinc massesin the center of each of the cells of the grid. The pattern can beletters printed from alternating conductive materials so that a messagecan be printed onto the primary surface-perhaps a brand name oridentifying information such as patient blood type.

Because the spontaneous oxidation-reduction reaction of silver and zincuses a ratio of approximately two silver to one zinc, the silver designcan contain about twice as much mass as the zinc design in anembodiment. At a spacing of about 1 mm between the closest dissimilarmetals (closest edge to closest edge) each voltaic cell that contacts aconductive fluid such as a cosmetic cream or anti-acne agent or skintreatment agent can create approximately 1 volt of potential that willpenetrate substantially through the dermis and epidermis. Closer spacingof the dots can decrease the resistance, providing less potential, andthe current will not penetrate as deeply. If the spacing falls belowabout one tenth of a millimeter a benefit of the spontaneous reaction isthat which is also present with a direct reaction; silver can beelectrically driven into the skin. Therefore, spacing between theclosest conductive materials can be 0.1 mm, or 0.2 mm, 0.3 mm, 0.4 mm,0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1 mm, 1.1 mm, 1.2 mm, 1.3 mm,1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm, 2 mm, 2.1 mm, 2.2 mm,2.3 mm, 2.4 mm, 2.5 mm, 2.6 mm, 2.7 mm, 2.8 mm, 2.9 mm, 3 mm, 3.1 mm,3.2 mm, 3.3 mm, 3.4 mm, 3.5 mm, 3.6 mm, 3.7 mm, 3.8 mm, 3.9 mm, 4 mm,4.1 mm, 4.2 mm, 4.3 mm, 4.4 mm, 4.5 mm, 4.6 mm, 4.7 mm, 4.8 mm, 4.9 mm,5 mm, 5.1 mm, 5.2 mm, 5.3 mm, 5.4 mm, 5.5 mm, 5.6 mm, 5.7 mm, 5.8 mm,5.9 mm, 6 mm, or the like.

In certain embodiments the spacing between the closest conductivematerials can be not more than 0.1 mm, or not more than 0.2 mm, not morethan 0.3 mm, not more than 0.4 mm, not more than 0.5 mm, not more than0.6 mm, not more than 0.7 mm, not more than 0.8 mm, not more than 0.9mm, not more than 1 mm, not more than 1.1 mm, not more than 1.2 mm, notmore than 1.3 mm, not more than 1.4 mm, not more than 1.5 mm, not morethan 1.6 mm, not more than 1.7 mm, not more than 1.8 mm, not more than1.9 mm, not more than 2 mm, not more than 2.1 mm, not more than 2.2 mm,not more than 2.3 mm, not more than 2.4 mm, not more than 2.5 mm, notmore than 2.6 mm, not more than 2.7 mm, not more than 2.8 mm, not morethan 2.9 mm, not more than 3 mm, not more than 3.1 mm, not more than 3.2mm, not more than 3.3 mm, not more than 3.4 mm, not more than 3.5 mm,not more than 3.6 mm, not more than 3.7 mm, not more than 3.8 mm, notmore than 3.9 mm, not more than 4 mm, not more than 4.1 mm, not morethan 4.2 mm, not more than 4.3 mm, not more than 4.4 mm, not more than4.5 mm, not more than 4.6 mm, not more than 4.7 mm, not more than 4.8mm, not more than 4.9 mm, not more than 5 mm, not more than 5.1 mm, notmore than 5.2 mm, not more than 5.3 mm, not more than 5.4 mm, not morethan 5.5 mm, not more than 5.6 mm, not more than 5.7 mm, not more than5.8 mm, not more than 5.9 mm, not more than 6 mm, or the like.

In certain embodiments spacing between the closest conductive materialscan be not less than 0.1 mm, or not less than 0.2 mm, not less than 0.3mm, not less than 0.4 mm, not less than 0.5 mm, not less than 0.6 mm,not less than 0.7 mm, not less than 0.8 mm, not less than 0.9 mm, notless than 1 mm, not less than 1.1 mm, not less than 1.2 mm, not lessthan 1.3 mm, not less than 1.4 mm, not less than 1.5 mm, not less than1.6 mm, not less than 1.7 mm, not less than 1.8 mm, not less than 1.9mm, not less than 2 mm, not less than 2.1 mm, not less than 2.2 mm, notless than 2.3 mm, not less than 2.4 mm, not less than 2.5 mm, not lessthan 2.6 mm, not less than 2.7 mm, not less than 2.8 mm, not less than2.9 mm, not less than 3 mm, not less than 3.1 mm, not less than 3.2 mm,not less than 3.3 mm, not less than 3.4 mm, not less than 3.5 mm, notless than 3.6 mm, not less than 3.7 mm, not less than 3.8 mm, not lessthan 3.9 mm, not less than 4 mm, not less than 4.1 mm, not less than 4.2mm, not less than 4.3 mm, not less than 4.4 mm, not less than 4.5 mm,not less than 4.6 mm, not less than 4.7 mm, not less than 4.8 mm, notless than 4.9 mm, not less than 5 mm, not less than 5.1 mm, not lessthan 5.2 mm, not less than 5.3 mm, not less than 5.4 mm, not less than5.5 mm, not less than 5.6 mm, not less than 5.7 mm, not less than 5.8mm, not less than 5.9 mm, not less than 6 mm, or the like.

Disclosed herein include LLEC or LLEF systems comprising a primarysurface of a pliable material wherein the pliable material is adapted tobe applied to an area of tissue such as the face of a subject; a firstelectrode design formed from a first conductive liquid that includes amixture of a polymer and a first element, the first conductive liquidbeing applied into a position of contact with the primary surface, thefirst element including a metal species, and the first electrode designincluding at least one dot or reservoir, wherein selective ones of theat least one dot or reservoir have approximately a 1.5 mm+/−1 mm meandiameter; a second electrode design formed from a second conductiveliquid that includes a mixture of a polymer and a second element, thesecond element including a different metal species than the firstelement, the second conductive liquid being printed into a position ofcontact with the primary surface, and the second electrode designincluding at least one other dot or reservoir, wherein selective ones ofthe at least one other dot or reservoir have approximately a 2.5 mm+/−2mm mean diameter; a spacing on the primary surface that is between thefirst electrode design and the second electrode design such that thefirst electrode design does not physically contact the second electrodedesign, wherein the spacing is approximately 1.5 mm+/−1 mm, and at leastone repetition of the first electrode design and the second electrodedesign, the at least one repetition of the first electrode design beingsubstantially adjacent the second electrode design, wherein the at leastone repetition of the first electrode design and the second electrodedesign, in conjunction with the spacing between the first electrodedesign and the second electrode design, defines at least one pattern ofat least one voltaic cell for spontaneously generating at least oneelectrical current when introduced to an electrolytic solution.Therefore, electrodes, dots or reservoirs can have a mean diameter of0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1.0 mm,1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm,2.0 mm, 2.1 mm, 2.2 mm, 2.3 mm, 2.4 mm, 2.5 mm, 2.6 mm, 2.7 mm, 2.8 mm,2.9 mm, 3.0 mm, 3.1 mm, 3.2 mm, 3.3 mm, 3.4 mm, 3.5 mm, 3.6 mm, 3.7 mm,3.8 mm, 3.9 mm, 4.0 mm, 4.1 mm, 4.2 mm, 4.3 mm, 4.4 mm, 4.5 mm, 4.6 mm,4.7 mm, 4.8 mm, 4.9 mm, 5.0 mm, or the like.

In further embodiments, electrodes, dots or reservoirs can have a meandiameter of not less than 0.2 mm, or not less than 0.3 mm, not less than0.4 mm, not less than 0.5 mm, not less than 0.6 mm, not less than 0.7mm, not less than 0.8 mm, not less than 0.9 mm, not less than 1.0 mm,not less than 1.1 mm, not less than 1.2 mm, not less than 1.3 mm, notless than 1.4 mm, not less than 1.5 mm, not less than 1.6 mm, not lessthan 1.7 mm, not less than 1.8 mm, not less than 1.9 mm, not less than2.0 mm, not less than 2.1 mm, not less than 2.2 mm, not less than 2.3mm, not less than 2.4 mm, not less than 2.5 mm, not less than 2.6 mm,not less than 2.7 mm, not less than 2.8 mm, not less than 2.9 mm, notless than 3.0 mm, not less than 3.1 mm, not less than 3.2 mm, not lessthan 3.3 mm, not less than 3.4 mm, not less than 3.5 mm, not less than3.6 mm, not less than 3.7 mm, not less than 3.8 mm, not less than 3.9mm, not less than 4.0 mm, not less than 4.1 mm, not less than 4.2 mm,not less than 4.3 mm, not less than 4.4 mm, not less than 4.5 mm, notless than 4.6 mm, not less than 4.7 mm, not less than 4.8 mm, not lessthan 4.9 mm, not less than 5.0 mm, or the like.

In further embodiments, electrodes, dots or reservoirs can have a meandiameter of not more than 0.2 mm, or not more than 0.3 mm, not more than0.4 mm, not more than 0.5 mm, not more than 0.6 mm, not more than 0.7mm, not more than 0.8 mm, not more than 0.9 mm, not more than 1.0 mm,not more than 1.1 mm, not more than 1.2 mm, not more than 1.3 mm, notmore than 1.4 mm, not more than 1.5 mm, not more than 1.6 mm, not morethan 1.7 mm, not more than 1.8 mm, not more than 1.9 mm, not more than2.0 mm, not more than 2.1 mm, not more than 2.2 mm, not more than 2.3mm, not more than 2.4 mm, not more than 2.5 mm, not more than 2.6 mm,not more than 2.7 mm, not more than 2.8 mm, not more than 2.9 mm, notmore than 3.0 mm, not more than 3.1 mm, not more than 3.2 mm, not morethan 3.3 mm, not more than 3.4 mm, not more than 3.5 mm, not more than3.6 mm, not more than 3.7 mm, not more than 3.8 mm, not more than 3.9mm, not more than 4.0 mm, not more than 4.1 mm, not more than 4.2 mm,not more than 4.3 mm, not more than 4.4 mm, not more than 4.5 mm, notmore than 4.6 mm, not more than 4.7 mm, not more than 4.8 mm, not morethan 4.9 mm, not more than 5.0 mm, or the like.

In embodiments, the density of the conductive materials can be, forexample, 20 reservoirs per square inch (/in²), 30 reservoirs/in², 40reservoirs/in², 50 reservoirs/in², 60 reservoirs/in², 70 reservoirs/in²,80 reservoirs/in², r 90 reservoirs/in², 100 reservoirs/in², 150reservoirs/in², 200 reservoirs/in², 250 reservoirs/in², 300reservoirs/in², or 350 reservoirs/in², 400 reservoirs/in², 450reservoirs/in², 500 reservoirs/in², 550 reservoirs/in², 600reservoirs/in², 650 reservoirs/in², 700 reservoirs/in², 750reservoirs/in², more, or the like.

In embodiments, the density of the conductive materials can be, forexample, more than 20 reservoirs/in², more than 30 reservoirs/in², morethan 40 reservoirs/in², more than 50 reservoirs/in², more than 60reservoirs/in², more than 70 reservoirs/in², more than 80reservoirs/in², more than 90 reservoirs/in², more than 100reservoirs/in², more than 150 reservoirs/in², more than 200reservoirs/in², more than 250 reservoirs/in², more than 300reservoirs/in², more than 350 reservoirs/in², more than 400reservoirs/in², more than 450 reservoirs/in², more than 500reservoirs/in², more than 550 reservoirs/in², more than 600reservoirs/in², more than 650 reservoirs/in², more than 700reservoirs/in², more than 750 reservoirs/in², or more, or the like.

The material concentrations or quantities within and/or the relativesizes (e.g., dimensions or surface area) of the first and secondreservoirs can be selected deliberately to achieve variouscharacteristics of the systems' behavior. For example, the quantities ofmaterial within a first and second reservoir can be selected to providean apparatus having an operational behavior that depletes atapproximately a desired rate and/or that “dies” after an approximateperiod of time after activation. In an embodiment the one or more firstreservoirs and the one or more second reservoirs are configured tosustain one or more currents for an approximate pre-determined period oftime, after activation. It is to be understood that the amount of timethat currents are sustained can depend on external conditions andfactors (e.g., the quantity and type of activation material), andcurrents can occur intermittently depending on the presence or absenceof activation material. Further disclosure relating to producingreservoirs that are configured to sustain one or more currents for anapproximate pre-determined period of time can be found in U.S. Pat. No.7,904,147 entitled SUBSTANTIALLY PLANAR ARTICLE AND METHODS OFMANUFACTURE issued Mar. 8, 2011, which is incorporated by referenceherein in its entirety.

In various embodiments the difference of the standard potentials of thefirst and second reservoirs can be in a range from 0.05 V toapproximately 5.0 V. For example, the standard potential can be 0.05 V,or 0.06 V, 0.07 V, 0.08 V, 0.09 V, 0.1 V, 0.2 V, 0.3 V, 0.4 V, 0.5 V,0.6 V, 0.7 V, 0.8 V, 0.9 V, 1.0 V, 1.1 V, 1.2 V, 1.3 V, 1.4 V, 1.5 V,1.6 V, 1.7 V, 1.8 V, 1.9 V, 2.0 V, 2.1 V, 2.2 V, 2.3 V, 2.4 V, 2.5 V,2.6 V, 2.7 V, 2.8 V, 2.9 V, 3.0 V, 3.1 V, 3.2 V, 3.3 V, 3.4 V, 3.5 V,3.6 V, 3.7 V, 3.8 V, 3.9 V, 4.0 V, 4.1 V, 4.2 V, 4.3 V, 4.4 V, 4.5 V,4.6 V, 4.7 V, 4.8 V, 4.9 V, 5.0 V, or the like.

In a particular embodiment the difference of the standard potentials ofthe first and second reservoirs can be at least 0.05 V, or at least 0.06V, at least 0.07 V, at least 0.08 V, at least 0.09 V, at least 0.1 V, atleast 0.2 V, at least 0.3 V, at least 0.4 V, at least 0.5 V, at least0.6 V, at least 0.7 V, at least 0.8 V, at least 0.9 V, at least 1.0 V,at least 1.1 V, at least 1.2 V, at least 1.3 V, at least 1.4 V, at least1.5 V, at least 1.6 V, at least 1.7 V, at least 1.8 V, at least 1.9 V,at least 2.0 V, at least 2.1 V, at least 2.2 V, at least 2.3 V, at least2.4 V, at least 2.5 V, at least 2.6 V, at least 2.7 V, at least 2.8 V,at least 2.9 V, at least 3.0 V, at least 3.1 V, at least 3.2 V, at least3.3 V, at least 3.4 V, at least 3.5 V, at least 3.6 V, at least 3.7 V,at least 3.8 V, at least 3.9 V, at least 4.0 V, at least 4.1 V, at least4.2 V, at least 4.3 V, at least 4.4 V, at least 4.5 V, at least 4.6 V,at least 4.7 V, at least 4.8 V, at least 4.9 V, at least 5.0 V, or thelike.

In a particular embodiment, the difference of the standard potentials ofthe first and second reservoirs can be not more than 0.05 V, or not morethan 0.06 V, not more than 0.07 V, not more than 0.08 V, not more than0.09 V, not more than 0.1 V, not more than 0.2 V, not more than 0.3 V,not more than 0.4 V, not more than 0.5 V, not more than 0.6 V, not morethan 0.7 V, not more than 0.8 V, not more than 0.9 V, not more than 1.0V, not more than 1.1 V, not more than 1.2 V, not more than 1.3 V, notmore than 1.4 V, not more than 1.5 V, not more than 1.6 V, not more than1.7 V, not more than 1.8 V, not more than 1.9 V, not more than 2.0 V,not more than 2.1 V, not more than 2.2 V, not more than 2.3 V, not morethan 2.4 V, not more than 2.5 V, not more than 2.6 V, not more than 2.7V, not more than 2.8 V, not more than 2.9 V, not more than 3.0 V, notmore than 3.1 V, not more than 3.2 V, not more than 3.3 V, not more than3.4 V, not more than 3.5 V, not more than 3.6 V, not more than 3.7 V,not more than 3.8 V, not more than 3.9 V, not more than 4.0 V, not morethan 4.1 V, not more than 4.2 V, not more than 4.3 V, not more than 4.4V, not more than 4.5 V, not more than 4.6 V, not more than 4.7 V, notmore than 4.8 V, not more than 4.9 V, not more than 5.0 V, or the like.In embodiments that include very small reservoirs (e.g., on thenanometer scale), the difference of the standard potentials can besubstantially less or more. The electrons that pass between the firstreservoir and the second reservoir can be generated as a result of thedifference of the standard potentials. Further disclosure relating tostandard potentials can be found in U.S. Pat. No. 8,224,439 entitledBATTERIES AND METHODS OF MANUFACTURE AND USE issued Jul. 17, 2012, whichis incorporated be reference herein in its entirety.

The voltage present at the site of acne treatment or skin rejuvenationis typically in the range of millivolts but disclosed embodiments canintroduce a much higher voltage, for example near 1 volt when using the1 mm spacing of dissimilar metals already described. The higher voltageis believed to drive the current deeper into the treatment area. In thisway the current not only can drive silver and zinc into the treatment ifdesired for treatment, but the current can also provide a stimulatorycurrent so that the entire surface area can be treated. The highervoltage may also increase antimicrobial effect bacteria and preventingbiofilms. The electric field can also have beneficial effects on cellmigration, ATP production, and angiogenesis.

While various embodiments have been shown and described, it will berealized that alterations and modifications can be made thereto withoutdeparting from the scope of the following claims. It is expected thatother methods of applying the conductive material can be substituted asappropriate. Also, there are numerous shapes, sizes and patterns ofvoltaic cells that have not been described but it is expected that thisdisclosure will enable those skilled in the art to incorporate their owndesigns which will then be applied to a surface to create voltaic cellswhich will become active when brought into contact with an electrolyticsolution.

Certain embodiments include LLEC or LLEF systems comprising embodimentsdesigned to be used on irregular, non-planar, or “stretching” surfaces.Embodiments disclosed herein can be used with numerous irregularsurfaces of the body, including the face, the shoulder, the elbow, thewrist, the finger joints, the hip, the knee, the ankle, the toe joints,etc. Additional embodiments disclosed herein can be used in areas wheretissue is prone to movement, for example the eyelid, the ear, the lips,the nose, the shoulders, the back, etc.

In certain embodiments, the substrate can be shaped to fit a particularregion of the body. As shown in FIG. 9, a mask-shaped substrate can beused for the treatment of acne around the face and forehead. Embodimentscan also include means for securing the mask to the user's head. In anembodiment the portion of the mask or substrate that is to contact theskin comprises a multi-array matrix of biocompatible microcells. Incertain embodiments a fluid or cream such as a conductive fluid or creamcan be applied between the multi-array matrix of biocompatiblemicrocells and the skin.

Embodiments can comprise a moisture-sensitive component that changescolor when the device is activated and producing an electric current.

Similarly, FIG. 13 depicts an embodiment designed to treat acne of theback. Water impermeable barrier 41 prevents or minimizes liquid fromescaping from the device. Hydration material 42 is between 41 and amulti-array matrix of biocompatible microcells layer 43. The hydrationmaterial 42 moistens layer 43. Adhesive velcro (or other suitableadhesion device) areas 44 affix layers 42 and 43 to layer 41. Edgingmaterial 45 is soft and flexible and protects the user from discomfort.Shoulder straps 46 are made of a soft, flexible material and ensure asecure fit on the patient. Soft, flexible waist strap 47 secures thelower portion of the embodiment to the patient.

Various apparatus embodiments which can be referred to as “medicalbatteries” are described herein. Further disclosure relating to thistechnology can be found in U.S. Pat. No. 7,672,719 entitled BATTERIESAND METHODS OF MANUFACTURE AND USE issued Mar. 2, 2010, which isincorporated herein by reference in its entirety.

Certain embodiments disclosed herein include a method of manufacturing asubstantially planar LLEC or LLEF system, the method comprising joiningwith a substrate multiple first reservoirs wherein selected ones of themultiple first reservoirs include a reducing agent, and wherein firstreservoir surfaces of selected ones of the multiple first reservoirs areproximate to a first substrate surface; and joining with the substratemultiple second reservoirs wherein selected ones of the multiple secondreservoirs include an oxidizing agent, and wherein second reservoirsurfaces of selected ones of the multiple second reservoirs areproximate to the first substrate surface, wherein joining the multiplefirst reservoirs and joining the multiple second reservoirs comprisesjoining using tattooing. In embodiments the substrate can comprisegauzes comprising dots or electrodes.

Further embodiments can include a method of manufacturing a LLEC or LLEFsystem, the method comprising joining with a substrate multiple firstreservoirs wherein selected ones of the multiple first reservoirsinclude a reducing agent, and wherein first reservoir surfaces ofselected ones of the multiple first reservoirs are proximate to a firstsubstrate surface; and joining with the substrate multiple secondreservoirs wherein selected ones of the multiple second reservoirsinclude an oxidizing agent, and wherein second reservoir surfaces ofselected ones of the multiple second reservoirs are proximate to thefirst substrate surface, wherein joining the multiple first reservoirsand joining the multiple second reservoirs comprises: combining themultiple first reservoirs, the multiple second reservoirs, and multipleparallel insulators to produce a pattern repeat arranged in a firstdirection across a plane, the pattern repeat including a sequence of afirst one of the parallel insulators, one of the multiple firstreservoirs, a second one of the parallel insulators, and one of themultiple second reservoirs; and weaving multiple transverse insulatorsthrough the first parallel insulator, the one first reservoir, thesecond parallel insulator, and the one second reservoir in a seconddirection across the plane to produce a woven apparatus.

Embodiments disclosed herein include LLEC and LLEF systems that canproduce an electrical stimulus and/or can electromotivate,electroconduct, electroinduct, electrotransport, and/or electrophoreseone or more therapeutic materials in areas of target tissue (e.g.,iontophoresis), and/or can cause one or more biologic or other materialsin proximity to, on or within target tissue to be rejuvenated. Furtherdisclosure relating to materials that can produce an electrical stimuluscan be found in U.S. Pat. No. 7,662,176 entitled FOOTWEAR APPARATUS ANDMETHODS OF MANUFACTURE AND USE issued Feb. 16, 2010, which isincorporated herein by reference in its entirety.

Embodiments disclosed herein include a multilayer fabric, for example alayer that can produce an LLEC/LLEF as described herein, a hydrationlayer, and a waterproof layer.

LLEC/LLEF Systems and Devices; Methods of Use

In embodiments, methods and devices disclosed herein can be used for totreat skin conditions. Examples of such treatments include the treatmentof acne, for example reducing or preventing the appearance of acne.Embodiments disclosed herein can comprise an acne treatment agent. Forexample, embodiments can comprise an acne treatment agent wherein theagent is located between the skin and the electrode surface. Examples ofsuch treatments also include the treatment of dark spots or patches onthe skin, or the treatment of wrinkles. Embodiments disclosed herein cancomprise a skin treatment agent. For example, embodiments can comprisean skin treatment agent wherein the agent is located between the skinand the electrode surface.

Embodiments disclosed herein relating to treatment of acne or skinrejuvenation can also comprise selecting a patient or tissue in need of,or that could benefit by, treatment for acne or skin rejuvenation.

Methods disclosed herein can include applying a disclosed embodiment toan area to be treated. Embodiments can include selecting or identifyinga patient in need of acne treatment or skin rejuvenation. Inembodiments, methods disclosed herein can include application of an acnetreatment agent to an area to be treated. In certain embodiments,disclosed methods include application of an acne treating agent to adevice disclosed herein.

In embodiments, methods and devices disclosed herein can be used toreduce the visibility of acne. In embodiments, methods and devicesdisclosed herein can be used to reduce the signs of skin aging. Thedevices can be used either alone or in conjunction with other componentswell known in the art, such as acne treatment agents or skinrejuvenation agents.

In embodiments, disclosed methods include application to the treatmentarea or the device an anti-aging or anti-acne and/or anti-rosacea activeagent. Examples of anti-acne and anti-rosacea agents include, but arenot limited to: retinoids such as tretinoin, isotretinoin, motretinide,adapalene, tazarotene, azelaic acid, and retinol; salicylic acid;benzoyl peroxide; resorcinol; sulfur; sulfacetamide; urea; antibioticssuch as tetracycline, clindamycin, metronidazole, and erythromycin;anti-inflammatory agents such as corticosteroids (e.g., hydrocortisone),ibuprofen, naproxen, and hetprofen; and imidazoles such as ketoconazoleand elubiol; and salts and prodrugs thereof. Other examples of anti-acneactive agents include essential oils, alpha-bisabolol, dipotassiumglycyrrhizinate, camphor, β-glucan, allantoin, feverfew, flavonoids suchas soy isoflavones, saw palmetto, chelating agents such as EDTA, lipaseinhibitors such as silver and copper ions, hydrolyzed vegetableproteins, inorganic ions of chloride, iodide, fluoride, and theirnonionic derivatives chlorine, iodine, fluorine, and syntheticphospholipids and natural phospholipids such as ARLASILK™.

In embodiments, disclosed methods include application to the treatmentarea of a cosmetic product, for example one that contains an anti-agingagent. Examples of suitable anti-aging agents include, but are notlimited to: inorganic sunscreens such as titanium dioxide and zincoxide; organic sunscreens such as octyl-methoxy cinnamates; retinoids;dimethylaminoathanol (DMAE), copper containing peptides, vitamins suchas vitamin E, vitamin A, vitamin C, and vitamin B and vitamin salts orderivatives such as ascorbic acid di-glucoside and vitamin E acetate orpalmitate; alpha hydroxy acids and their precursors such as glycolicacid, citric acid, lactic acid, malic acid, mandelic acid, ascorbicacid, alpha-hydroxybutyric acid, alpha-hydroxyisobutyric acid,alpha-hydroxyisocaproic acid, atrrolactic acid, alpha-hydroxyisovalericacid, ethyl pyruvate, galacturonic acid, glucoheptonic acid,glucoheptono 1,4-lactone, gluconic acid, gluconolactone, glucuronicacid, glucuronolactone, isopropyl pyruvate, methyl pyruvate, mucic acid,pyruvic acid, saccharic acid, saccaric acid 1,4-lactone, tartaric acid,and tartronic acid; beta hydroxy acids such as beta-hydroxybutyric acid,beta-phenyl-lactic acid, and beta-phenylpyruvic acid; tetrahydroxypropylethylenediamine, N,N,N′,N′-Tetrakis(2-hydroxypropyl)ethylenediamine(THPED); and botanical extracts such as green tea, soy, milk thistle,algae, aloe, angelica, bitter orange, coffee, goldthread, grapefruit,hoellen, honeysuckle, Job's tears, lithospermum, mulberry, peony,puerarua, nice, and safflower; and salts and prodrugs thereof

In embodiments, the anti-acne agent or anti-aging agent or skinrejuvenation agent contains a depigmentation agent. Examples of suitabledepigmentation agents include, but are not limited to: soy extract; soyisoflavones; retinoids such as retinol; kojic acid; kojic dipalmitate;hydroquinone; arbutin; transexamic acid; vitamins such as niacin andvitamin C; azelaic acid; linolenic acid and linoleic acid; placertia;licorice; and extracts such as chamomile and green tea; and salts andprodrugs thereof.

In an exemplary embodiment, a method disclosed herein comprises applyinga conductive acne treatment or anti-acne agent to an area wheretreatment is desired, then applying over the agent a bioelectric devicethat comprises a multi-array matrix of biocompatible microcells.

In an exemplary embodiment, a method disclosed herein comprises applyinga conductive skin rejuvenation agent to an area where treatment isdesired, then applying over the agent a bioelectric device thatcomprises a multi-array matrix of biocompatible microcells.

EXAMPLES

The following non-limiting examples are provided for illustrativepurposes only in order to facilitate a more complete understanding ofrepresentative embodiments. These examples should not be construed tolimit any of the embodiments described in the present specificationincluding those pertaining to the methods of treating acne.

Example 1 Cell Migration Assay

The in vitro scratch assay is an easy, low-cost and well-developedmethod to measure cell migration in vitro. The basic steps involvecreating a “scratch” in a cell monolayer, capturing images at thebeginning and at regular intervals during cell migration to close thescratch, and comparing the images to quantify the migration rate of thecells. Compared to other methods, the in vitro scratch assay isparticularly suitable for studies on the effects of cell-matrix andcell-cell interactions on cell migration, mimic cell migration duringwound healing in vivo and are compatible with imaging of live cellsduring migration to monitor intracellular events if desired. In additionto monitoring migration of homogenous cell populations, this method hasalso been adopted to measure migration of individual cells in theleading edge of the scratch. Not taking into account the time fortransfection of cells, in vitro scratch assay per se usually takes fromseveral hours to overnight.

Human keratinocytes were plated under plated under placebo or a LLECsystem (labeled “PROCELLERA®”). Cells were also plated under silver-onlyor zinc-only dressings. After 24 hours, the scratch assay was performed.Cells plated under the PROCELLERA® device displayed increased migrationinto the “scratched” area as compared to any of the zinc, silver, orplacebo dressings. After 9 hours, the cells plated under the PROCELLERA®device had almost “closed” the scratch. This demonstrates the importanceof electrical activity to cell migration and infiltration.

In addition to the scratch test, genetic expression was tested.Increased insulin growth factor (IGF)-1R phosphorylation wasdemonstrated by the cells plated under the PROCELLERA® device ascompared to cells plated under insulin growth factor alone.

Integrin accumulation also affects cell migration. An increase inintegrin accumulation achieved with the LLEC system. Integrin isnecessary for cell migration, and is found on the leading edge ofmigrating cell.

Thus, the tested LLEC system enhanced cellular migration andIGF-1R/integrin involvement. This involvement demonstrates the effectthat the LLEC system had upon cell receptors involved with the woundhealing process.

Example 2 Zone of Inhibition Test

For cellular repair to be most efficient, available energy should not beshared with ubiquitous microbes. In this “zone of inhibition” test,placebo, a LLEC device (PROCELLERA®) and silver only were tested in anagar medium with a 24 hour growth of organisms. Bacterial growth waspresent over the placebo, a zone of inhibition over the PROCELLERA® anda minimal inhibition zone over the silver. Because the samples were“buried” in agar, the electricidal effect of the LLEC system could betested. Silver ion diffusion, the method used by silver basedantimicrobials, alone was not sufficient. The test demonstrates theimproved bactericidal effect of PROCELLERA® as compared to silver alone.

Example 3 LLEC Influence on Human Keratinocyte Migration

An LLEC-generated electrical field was mapped, leading to theobservation that LLEC generates hydrogen peroxide, known to drive redoxsignaling. LLEC-induced phosphorylation of redox-sensitive IGF-1R wasdirectly implicated in cell migration. The LLEC also increasedkeratinocyte mitochondrial membrane potential.

The LLEC was made of polyester printed with dissimilar elemental metalsas described herein. It comprises alternating circular regions of silverand zinc dots, along with a proprietary, biocompatible binder added tolock the electrodes to the surface of a flexible substrate in a patternof discrete reservoirs. When the LLEC contacts an aqueous solution, thesilver positive electrode (cathode) is reduced while the zinc negativeelectrode (anode) is oxidized. The LLEC used herein consisted of metalsplaced in proximity of about 1 mm to each other thus forming a redoxcouple and generating an ideal potential on the order of 1 Volt. Thecalculated values of the electric field from the LLEC were consistentwith the magnitudes that are typically applied (1-10 V/cm) in classicalelectrotaxis experiments, suggesting that cell migration observed withthe bioelectric dressing is likely due to electrotaxis.

Measurement of the potential difference between adjacent zinc and silverdots when the LLEC is in contact with de-ionized water yielded a valueof about 0.2 Volts. Though the potential difference between zinc andsilver dots can be measured, non-intrusive measurement of the electricfield arising from contact between the LLEC and liquid medium wasdifficult. Keratinocyte migration was accelerated by exposure to anAg/Zn LLEC. Replacing the Ag/Zn redox couple with Ag or Zn alone did notreproduce the effect of keratinocyte acceleration.

Exposing keratinocytes to a LLEC for 24 h significantly increased greenfluorescence in the dichlorofluorescein (DCF) assay indicatinggeneration of reactive oxygen species under the effect of the LLEC. Todetermine whether H₂O₂ is generated specifically, keratinocytes werecultured with a LLEC or placebo for 24 h and then loaded with PF6-AM(Peroxyfluor-6 acetoxymethyl ester; an indicator of endogenous H₂O₂).Greater intracellular fluorescence was observed in the LLECkeratinocytes compared to the cells grown with placebo. Over-expressionof catalase (an enzyme that breaks down H₂O₂) attenuated the increasedmigration triggered by the LLEC. Treating keratinocytes with N-AcetylCysteine (which blocks oxidant-induced signaling) also failed toreproduce the increased migration observed with LLEC. Thus, H₂O₂signaling mediated the increase of keratinocyte migration under theeffect of the electrical stimulus.

External electrical stimulus can up-regulate the TCA (tricarboxylicacid) cycle. The stimulated TCA cycle is then expected to generate moreNADH and FADH₂ to enter into the electron transport chain and elevatethe mitochondrial membrane potential (Δm). Fluorescent dyes JC-1 andTMRM were used to measure mitochondrial membrane potential. JC-1 is alipophilic dye which produces a red fluorescence with high Δm and greenfluorescence when Δm is low. TMRM produces a red fluorescenceproportional to Δm. Treatment of keratinocytes with LLEC for 24 hdemonstrated significantly high red fluorescence with both JC-1 andTMRM, indicating an increase in mitochondrial membrane potential andenergized mitochondria under the effect of the LLEC. As a potentialconsequence of a stimulated TCA cycle, available pyruvate (the primarysubstrate for the TCA cycle) is depleted resulting in an enhanced rateof glycolysis. This can lead to an increase in glucose uptake in orderto push the glycolytic pathway forward. The rate of glucose uptake inHaCaT cells treated with LLEC was examined next. More than two foldenhancement of basal glucose uptake was observed after treatment withLLEC for 24 h as compared to placebo control.

Keratinocyte migration is known to involve phosphorylation of a numberof receptor tyrosine kinases (RTKs). To determine which RTKs areactivated as a result of LLEC, scratch assay was performed onkeratinocytes treated with LLEC or placebo for 24 h. Samples werecollected after 3 h and an antibody array that allows simultaneousassessment of the phosphorylation status of 42 RTKs was used to quantifyRTK phosphorylation. It was determined that LLEC significantly inducesIGF-1R phosphorylation. Sandwich ELISA using an antibody againstphospho-IGF-1R and total IGF-1R verified this determination. As observedwith the RTK array screening, potent induction in phosphorylation ofIGF-1R was observed 3 h post scratch under the influence of LLEC. IGF-1Rinhibitor attenuated the increased keratinocyte migration observed withLLEC treatment.

MBB (monobromobimane) alkylates thiol groups, displacing the bromine andadding a fluorescent tag (lamda emission=478 nm). MCB (monochlorobimane)reacts with only low molecular weight thiols such as glutathione.Fluorescence emission from UV laser-excited keratinocytes loaded witheither MBB or MCB was determined for 30 min. Mean fluorescence collectedfrom 10,000 cells showed a significant shift of MBB fluorescenceemission from cells. No significant change in MCB fluorescence wasobserved, indicating a change in total protein thiol but notglutathione. HaCaT cells were treated with LLEC for 24 h followed by ascratch assay. Integrin expression was observed by immuno-cytochemistryat different time points. Higher integrin expression was observed 6 hpost scratch at the migrating edge.

Consistent with evidence that cell migration requires H₂O₂ sensing, wedetermined that by blocking H₂O₂ signaling by decomposition of H₂O₂ bycatalase or ROS scavenger, N-acetyl cysteine, the increase inLLEC-driven cell migration is prevented. The observation that the LLECincreases H₂O₂ production is significant because in addition to cellmigration, hydrogen peroxide generated in the wound margin tissue isrequired to recruit neutrophils and other leukocytes to the wound,regulates monocyte function, and VEGF signaling pathway and tissuevascularization. Therefore, external electrical stimulation can be usedas an effective strategy to deliver low levels of hydrogen peroxide overtime to mimic the environment of the healing wound and thus should helpimprove wound outcomes. Another phenomenon observed duringre-epithelialization is increased expression of the integrin subunit αv.There is evidence that integrin, a major extracellular matrix receptor,polarizes in response to applied ES and thus controls directional cellmigration. It may be noted that there are a number of integrin subunits,however we chose integrin αv because of evidence of association of αvintegrin with IGF-1R, modulation of IGF-1 receptor signaling, and ofdriving keratinocyte locomotion. Additionally, integrin_(αv) has beenreported to contain vicinal thiols that provide site for redoxactivation of function of these integrins and therefore the increase inprotein thiols that we observe under the effect of ES may be the drivingforce behind increased integrin mediated cell migration. Other possibleintegrins which may be playing a role in LLEC-induced IGF-1R mediatedkeratinocyte migration are α5 integrin and α6 integrin.

Materials and Methods

Cell culture—Immortalized HaCaT human keratinocytes were grown inDulbecco's low-glucose modified Eagle's medium (Life Technologies,Gaithersburg, Md., U.S.A.) supplemented with 10% fetal bovine serum, 100U/ml penicillin, and 100 μg/ml streptomycin. The cells were maintainedin a standard culture incubator with humidified air containing 5% CO₂ at37° C.

Scratch assay—A cell migration assay was performed using culture inserts(IBIDI®, Verona, Wis.) according to the manufacturers instructions. Cellmigration was measured using time-lapse phase-contrast microscopyfollowing withdrawal of the insert. Images were analyzed using theAxioVision Rel 4.8 software.

N-Acetyl Cysteine Treatment—Cells were pretreated with 5 mM of the thiolantioxidant N-acetylcysteine (Sigma) for 1 h before start of the scratchassay.

IGF-1R inhibition—When applicable, cells were preincubated with 50 nMIGF-1R inhibitor, picropodophyllin (Calbiochem, MA) just prior to theScratch Assay.

Cellular H₂O₂ Analysis—To determine intracellular H₂O₂ levels, HaCaTcells were incubated with 5 pM PF6-AM in PBS for 20 min at roomtemperature. After loading, cells were washed twice to remove excess dyeand visualized using a Zeiss Axiovert 200M microscope.

Catalase gene delivery—HaCaT cells were transfected with 2.3×10⁷ pfuAdCatalase or with the empty vector as control in 750 μL of media.Subsequently, 750 μL of additional media was added 4 h later and thecells were incubated for 72 h.

RTK Phosphorylation Assay—Human Phospho-Receptor Tyrosine Kinasephosphorylation was measured using Phospho-RTK Array kit (R & DSystems).

ELISA—Phosphorylated and total IGF-1R were measured using a DuoSet ICELISA kit from R&D Systems.

Determination of Mitochondrial Membrane Potential—Mitochondrial membranepotential was measured in HaCaT cells exposed to the LLEC or placebousing TMRM or JC-1 (MitoProbe JC-1 Assay Kit for Flow Cytometry, LifeTechnologies), per manufacturers instructions for flow cytometry.

Integrin αV Expression—Human HaCaT cells were grown under the MCD orplacebo and harvested 6 h after removing the IBIDI® insert. Staining wasdone using antibody against integrin αV (Abcam, Cambridge, Mass.).

Example 4 Wound Care Study

The medical histories of patients who received “standard-of-care” woundtreatment (“SOC”; n=20), or treatment with a LLEC device as disclosedherein (n=18), were reviewed. The wound care device used in the presentstudy consisted of a discrete matrix of silver and zinc dots. Asustained voltage of approximately 0.8 V was generated between the dots.The electric field generated at the device surface was measured to be0.2-1.0 V, 10-50 μA.

Wounds were assessed until closed or healed. The number of days to woundclosure and the rate of wound volume reduction were compared. Patientstreated with LLEC received one application of the device each week, ormore frequently in the presence of excessive wound exudate, inconjunction with appropriate wound care management. The LLEC was keptmoist by saturating with normal saline or conductive hydrogel.Adjunctive therapies (such as negative pressure wound therapy [NPWT],etc.) were administered with SOC or with the use of LLEC unlesscontraindicated. The SOC group received the standard of care appropriateto the wound, for example antimicrobial dressings, barrier creams,alginates, silver dressings, absorptive foam dressings, hydrogel,enzymatic debridement ointment, NPWT, etc. Etiology-specific care wasadministered on a case-by-case basis. Dressings were applied at weeklyintervals or more. The SOC and LLEC groups did not differ significantlyin gender, age, wound types or the length, width, and area of theirwounds.

Wound dimensions were recorded at the beginning of the treatment, aswell as interim and final patient visits. Wound dimensions, includinglength (L), width (W) and depth (D) were measured, with depth measuredat the deepest point. Wound closure progression was also documentedthrough digital photography. Determining the area of the wound wasperformed using the length and width measurements of the wound surfacearea.

Closure was defined as 100% epithelialization with visible effacement ofthe wound. Wounds were assessed 1 week post-closure to ensure continuedprogress toward healing during its maturation and remodeling phase.

Wound types included in this study were diverse in etiology anddimensions, thus the time to heal for wounds was distributed over a widerange (9-124 days for SOC, and 3-44 days for the LLEC group).Additionally, the patients often had multiple co-morbidities, includingdiabetes, renal disease, and hypertension. The average number of days towound closure was 36.25 (SD=28.89) for the SOC group and 19.78(SD=14.45) for the LLEC group, p=0.036. On average, the wounds in theLLEC treatment group attained closure 45.43% earlier than those in theSOC group.

Based on the volume calculated, some wounds improved persistently whileothers first increased in size before improving. The SOC and the LLECgroups were compared to each other in terms of the number of instanceswhen the dimensions of the patient wounds increased (i.e., woundtreatment outcome degraded). In the SOC group, 10 wounds (50% for n=20)became larger during at least one measurement interval, whereas 3 wounds(16.7% for n=18) became larger in the LLEC group (p=0.018). Overall,wounds in both groups responded positively. Response to treatment wasobserved to be slower during the initial phase, but was observed toimprove as time progressed.

The LLEC wound treatment group demonstrated on average a 45.4% fasterclosure rate as compared to the SOC group. Wounds receiving SOC weremore likely to follow a “waxing-and-waning” progression in wound closurecompared to wounds in the LLEC treatment group.

Compared to localized SOC treatments for wounds, the LLEC (1) reduceswound closure time, (2) has a steeper wound closure trajectory, and (3)has a more robust wound healing trend with fewer incidence of increasedwound dimensions during the course of healing.

Example 5 Induction of Pre-Angiogenic Responses in Vascular EndothelialCells by Signaling Through VEGF Receptors

Materials and Methods

Cell Cultures and Reagents

Tissue culture reagents were obtained from Life Technologies UK. TheVEGFR inhibitor (catalog number 676475), the PI3K inhibitor LY294002(catalog number 440202), the Akt inhibitor (catalog number 124005) andthe Rho kinase inhibitor Y27632 (catalog number 688001) were allobtained from Calbiochem. Rhodamine-phalloidin (E3478) was obtained fromMolecular Probes (Leiden, The Netherlands) and anti-tubulin conjugatedwith FITC was obtained from Sigma. The HUVEC cell line from ATCC wasused prior to passage 10. Dulbecco's modified Eagle's medium (DMEM) with10% fetal bovine serum (FBS) was used for culture cells and EF exposureexperiments.

Electric Field Stimulation

HUVEC cells were seeded in a trough formed by two parallel (1 cm apart)strips of glass coverslip (No. 1, length of 22 mm) fixed to the base ofthe dish with silicone grease. Scratch lines were made perpendicular tothe long axis of the chamber with a fine sterile needle and used asreference marks for directed cell migration. Cells were incubated for24-48 hours (37° C., 5% CO₂) before a roof coverslip was applied andsealed with silicone grease. The final dimensions of the chamber,through which current was passed, were 22×10×0.2 mm. Agar-salt bridgesnot less than 15 cm long were used to connect silver/silver-chlorideelectrodes in beakers of Steinberg's solution (58 mM NaCl, 0.67 mM KCl,0.44 mM Ca(NO₃)₂, 1.3 mM MgSO₄, 4.6 mM Trizma base, pH 7.8-8.0), topools of excess culture medium at either side of the chamber. Fieldstrengths were measured directly at the beginning of, the end of andduring each experiment. No fluctuations in field strength were observed.For drug inhibition experiments, cells were incubated with the VEGFRinhibitor 4-[(4′-chloro-2′-fluoro)phenylamino]-6,7-dimethoxyquinazoline(50 μM), the PI3K inhibitor LY294002 (50 μM), an Akt inhibitor1-L-6-hydroxymethyl-chiro-inositol2-[(R)-2-O-methyl-3-O-octadecylcarbonate] (50 μM), the Rho kinaseinhibitor Y27632 (50 μM), both Akt and Rho kinase inhibitors (10 μMeach) or latrunculin (50 nM) for 1 hour before EF stimulation. The sameconcentration of drug was present during EF exposure in a CO₂ incubator.

Quantification of Cell Behavior

A series of images was taken with an image analyser immediately beforeEF exposure and at 4, 8 and 24 hours of EF exposure. Cell orientationwas quantified as an orientation index (Oi), which is defined as Oi=cos2(α), where α is the angle formed by the long axis of a cell with a linedrawn perpendicular to the field lines. A cell with its long axisparallel to the vector of the EF will have an Oi of −1, and a cell withits long axis exactly perpendicular to the EF vector will have an Oi of+1. A randomly oriented population of cells will have an average Oi{defined as [Σ_(n) cos 2(α)]÷n} of 0. The significance of thistwo-dimensional orientation distribution against randomness wascalculated using Rayleigh's distribution. A long:short axis ratio wascalculated for assessment of elongation.

Mean migration rate and directedness were quantified over 4 hoursbecause cells multiplied during longer EF exposures, making it difficultto define a clear migration path. The angle (θ) that each cell movedwith respect to the imposed EF vector was measured. The cos(θ)(directedness) is +1, if the cell moved directly along the field linestoward the cathode, 0 if the cell moved perpendicular to the EF vectorand −1 if the cell moved directly towards the positive pole. Averagingthe cosines {[Σ_(i) cos(θ)]÷N, where N is the total number of cells}yields an average directedness of cell movement.

A commercially available VEGF165 ELISA kit was obtained from R and D(Minneapolis, Minn.), and the detailed technical instructions werefollowed. Confocal microscopy was as described. Statistical analyseswere performed using unpaired, two-tailed Student's t-test. Data areexpressed as mean±s.e.m.

Results

Cells cultured without exposure to the EF had the typical cobblestonemorphology, with the long axis of the cell body oriented randomly. Incontrast, endothelial cells cultured in DC EFs underwent areorientation, with their long axis coming to lie perpendicular to thevector of the applied EF. This elongation and alignment in an applied EFresembles the response of endothelial cells to fluid shear stress.

Cell alignment was quantified using an orientation index Oi=cos 2(α),where α is the angle formed between the long axis of a cell and a linedrawn perpendicular to the field lines. In cells oriented perpendicularto the field vector, the Oi is +1, cells parallel to the field vectorgive an Oi of −1 and random orientation gives an Oi of 0. We comparedthe elongation and reorientation of single cells with those of cells inmonolayers. They were broadly similar, with single cells respondingquicker and showing a significantly higher Oi (0.56±0.04, n=245) at 4hours of EF exposure than cells in a monolayer sheet (0.35±0.03, n=528).Both single cells and cells in monolayers, however, had a similar Oi by8 hours (0.71±0.03, n=227 and 0.62±0.03, n=312, respectively).

The perpendicular orientation of endothelial cells showed both time andvoltage dependency. Significant orientation was observed as early as 4hours after the onset of the EF. A steady increase of Oi indicatesgradually increasing perpendicular orientation with continued exposure.Longer EF exposure, up to 3 days at 100 mV mm⁻¹ (1 mV across a cell 10μm wide), induced striking orientation and elongation. EF exposure didnot induce any detrimental effects on the cells, which were perfectlyhealthy for up to 3-4 days in EFs.

Voltage dependency was more obvious at later times, with a higher Oi forcells cultured at higher voltages. After 24 hours at 300 mV mm⁻¹, almostall the cells were perpendicular. An EF strength as low as 75 mV mm⁻¹induced significant perpendicular orientation, with Oi of 0.19(significantly different from random orientation, p=4.4×10⁻⁶, n=433),whereas an EF of 50 mV mm⁻¹ did not. The threshold field strengthinducing perpendicular orientation of the endothelial cells wastherefore between 50 mV mm⁻¹ and 75 mV mm⁻¹. This is low, representingonly 0.5-0.75 mV across a cell with a diameter of 10 μm.

Reorientation of Endothelial Cells in EFs Requires VEGFR Activation

VEGF activation is a pivotal element in angiogenic responses andenhanced angiogenesis by electric stimulation in vivo is mediatedthrough VEGFR activation. To test whether EF-induced endothelial cellorientation might involve VEGF signaling, we quantified levels of VEGF.EF exposure (200 mV mm⁻¹, the same as that measured at skin wounds)significantly enhanced levels of VEGF released into the culture medium.Marked elevation of VEGF in the culture medium was observed as early as5 minutes after onset of the EF; this was reduced at 1 hour and 2 hours,rose again at 4 hours, and reached a high level by 24 hours.

Inhibition of VEGFR activation by inhibiting both VEGFR-1 and VEGFR-2with the drug4-[(4′-chloro-2′-fluoro)phenylamino]-6,7-dimethoxyquinazoline completelyabolished the reorientation of cells in an EF. This drug is a potentVEGFR inhibitor that inhibits the receptor tyrosine kinase activity (50%inhibitory concentrations of 2.0 μM and 100 nM for VEGFR-1 and VEGFR-2,respectively). It is very selective for VEGFR-1 and VEGFR-2 tyrosinekinase activity compared with that associated with the epidermal growthfactor (EGF) receptor (50-fold and 3800-fold, respectively). Themorphology of the cells treated with VEGFR inhibitor was very similar tocontrol cells. Cells still elongated, although their long axis wasslightly reduced, but they were oriented randomly. Inhibition of VEGFRscould conceivably have had detrimental effects on the long-termviability of cells and this could have influenced their orientationresponses. To test for this, we compared the orientation response aftera short period of inhibitor and EF application. The orientation responsewas completely abolished at 4 hours and 8 hours in an EF after VEGFRinhibition. The Oi values of the cells treated with VEGFR inhibitor were−0.16±0.05 and −0.05±0.05 in EF for 4 hours and 8 hours, respectively,which is significantly different from the non-inhibitor-treated valuesof 0.36±0.05 and 0.53±0.05 (P<0.01).

Reorientation of Endothelial Cells Involved the PI3K-Akt Pathway

VEGFR activation lead to endothelial cell migration, cell survival andproliferation, which require the activation of Akt, a downstreameffectors of PI3K. Both the PI3K inhibitor LY294002 (50 μM) and the Aktinhibitor (50 μM) significantly decreased the orientation response.

The concentration of either drug alone would be expected to inhibit PI3Kand Akt activation completely but neither drug inhibited perpendicularreorientation completely, and significant Oi values remained, indicatingthat other signaling mechanisms must be involved.

Role of Rho and Integrin in EF-Induced Reorientation of EndothelialCells

The Rho family of GTPases regulates VEGF-stimulated endothelial cellmotility and reorganization of the actin cytoskeleton, which areimportant in endothelial cell retraction and in the formation ofintercellular gaps. The Rho kinase inhibitor, Y27632, decreased theorientation response significantly, with Oi values of 0.55±0.05,0.45±0.05 and 0.24±0.05 at 10 μM, 20 μM and 50 μM, respectively.Significant Oi values nonetheless remained even at 50 μM, indicatingthat multiple signaling mechanisms must be involved.Mitogen-activated-protein kinase inhibition with U0126 (50 μM), likeY27632 (0.33±0.03), decreased the orientation to a similar extent.

Because both Akt and Rho kinase inhibitors individually showed partialinhibition, perhaps the two enzymes function in different pathways toinduce cell reorientation. To test this, a combination of the twoinhibitors was used. The orientation response was abolished completelyby using Akt and Rho kinase inhibitors together (both at 10 μM)(Oi=−0.10±0.06; compared to control=0.80±0.09, P<0.0001).

Integrins, especially αvβ3, are important in endothelial cell movementand alignment to shear stress and mechanical stimulation. HUVEC cellswere incubated with a blocking antibody against αvβ3 (LM609) (20 μgml⁻¹) for 1 hour and then exposed to an EF (200 mV mm⁻¹) with theantibody present. Blocking αvβ3 had no effect on orientation to the EF,cells reoriented normally (Oi=0.72±0.03, n=110, compared with thecontrol=0.80±0.09, n=124, P>0.05).

Small EFs Elongated Endothelial Cells

HUVEC cells elongated dramatically in an EF. By contrast, cells culturedwith no EF retained a more-cobblestone-like appearance. Striking cellelongation was induced by a voltage drop of about 0.7-4.0 mV across acell of ˜15 μm in diameter. We quantified the elongation of the cellsusing a long:short axis ratio. A perfectly round cell has a long:shortaxis ratio of 1 and, as cells elongate, the ratio increases. Controlcells (no EF) showed no increase in long:short axis over 24 hours inculture. Elongation responses were both time and voltage dependent. Thelong:short axis ratio of EF exposed cells indicated gradual cellelongation throughout the 24 hour experimental period. The voltagedependency of the elongation response was more obvious at later times,with a greater long:short axis ratio for cells cultured at higher EFs.The threshold for EF-induced endothelial cell elongation was between50-75 mV mm⁻¹, again 0.5-0.75 mV across a cell 10 μm in diameter. Theelongation response of endothelial cells was more marked than that seenpreviously at the same EF strengths, in corneal and lens epithelialcells.

VEGFR, PI3K-Akt and Rho Signaling are Involved in the ElongationResponse

The signaling elements required for reorientation are also involved inelongation, but there are subtle differences. The VEGFR inhibitor (50μM) had no effect on the long:short axis ratio of control cells butsignificantly decreased the long:short axis ratio in EF-treated cells(P<0.002). Both the PI3K inhibitor LY294002 and the Akt inhibitor alsosignificantly decreased the long:short axis ratio (both P<0.0001 versuscontrol). Cells treated with these drugs elongated less, with LY294002the more effective in suppressing EF-induced elongation. The Rho kinaseinhibitor, Y27632 also significantly decreased the long:short axis ratio(P<0.0001), whereas the αvβ3-blocking antibody significantly inhibitedthe elongation response (3.12±0.008 compared with the control 3.65±0.15,P=0.007).

Cytoskeleton Alignment and the Consequence of Actin Filament Disruption

To control changes in cell shape, reorientation and migration,extracellular stimuli initiate intracellular signaling that modifiescytoskeletal organization. Both actin filaments and microtubules werealigned in the direction of cell elongation. Latrunculin A, a toxininhibiting actin polymerization, completely abolished the EF-inducedelongation response and suppressed the orientation responsesignificantly (P<0.001) but not fully.

Small EFs Direct Migration of Endothelial Cells Towards the Anode

Endothelial cells migrated directionally toward the anode when culturedin EFs. The directional migration was slow but steady during the EFexposure and was more evident for single cells than for sheets of cells.Cells migrated directionally towards the anode while elongating andreorienting perpendicularly. Lamellipodial extension toward the anodewas marked. Directional migration was obvious at a physiological EFstrength of 100 mV mm⁻¹. The threshold field strength that could inducedirectional migration was therefore below 100 mV mm⁻¹. Cell migrationwas quantified as previously and significant anodal migration wasevident (P<0.0001). Migration speed, however, remained constant beforeand after EF exposure, at 1-2 μm hour⁻¹, which is significantly slowerthan most other cell types migrating in an EF.

Example 6 Effect on Propionibacterium acnes

Bacterial Strains and Culture

The main bacterial strain used in this study is Propionibacterium acnesand multiple antibiotics-resistant P. acnes isolates are to beevaluated.

ATCC medium (7 Actinomyces broth) (BD) and/or ATCC medium (593 choppedmeat medium) is used for culturing P. acnes under an anaerobic conditionat 37° C. All experiments are performed under anaerobic conditions.

Culture

LNA (Leeming-Notman agar) medium is prepared and cultured at 34° C. for14 days.

Planktonic Cells

P. acnes is a relatively slow-growing, typically aero-tolerantanaerobic, Gram-positive bacterium (rod). P. acnes is cultured underanaerobic condition to determine for efficacy of an embodiment disclosedherein (PROCELLERA®). Overnight bacterial cultures are diluted withfresh culture medium supplemented with 0.1% sodium thioglycolate in PBSto 10⁵ colony forming units (CFUs). Next, the bacterial suspensions (0.5mL of about 105) are applied directly on PROCELLERA® (2″×2″) and controlfabrics in Petri-dishes under anaerobic conditions. After 0 h and 24 hpost treatments at 37° C., portions of the sample fabrics are placedinto anaerobic diluents and vigorously shaken by vortexing for 2 min.The suspensions are diluted serially and plated onto anaerobic platesunder an anaerobic condition. After 24 h incubation, the survivingcolonies are counted.

Bacterial Biofilms in Skin Infections

It is generally accepted that many human infections are biofilm-relatedand that sessile (biofilm-grown) cells are highly resistant againstantimicrobial agents. It has been suggested that P. acnes cells residingwithin the follicles grow as a biofilm. P. acnes readily forms biofilmsin vitro as well as on various medical devices in vivo, combined withthe high resistance of sessile P. acnes cells and the increasedproduction of particular virulence factors.

Example 7 Reduction of Facial Wrinkles

A 44 year old male seeks treatment for wrinkles around his eyes (crows'feet). The patient's face is imaged in 3 dimensions and from this data amask with a multi-array matrix of biocompatible microcells is producedfrom a pliable material. Before going to sleep, the patient appliesEstee Lauder Advanced Night Repair as directed. The patient then puts onthe mask so that the multi-array matrix of biocompatible microcellscontacts the cosmetic product which contacts his skin. The multi-arraymatrix of produces a LLEC when it contacts the cosmetic product. Thepatient repeats this each night. After 30 days of treatment thepatient's crows' feet are visibly reduced.

Example 8 Reduction of Facial Wrinkles

A 50 year old female seeks treatment for wrinkles on her face. Thepatient's face is imaged in 3 dimensions and from this data a mask witha multi-array matrix of biocompatible microcells is produced from apliable material. Before going to sleep, the patient applies a liquidcosmetic product to her face. The patient then puts on the mask so thatthe multi-array matrix of biocompatible microcells contacts the cosmeticproduct which contacts her skin. The multi-array matrix of biocompatiblemicrocells produces a LLEC when it contacts the cosmetic product. Themask is worn until the patient awakes. The patient repeats this eachnight. After 20 days of treatment the wrinkles are visibly reduced.

Example 9 Reduction of Facial Wrinkles

A 38 year old male seeks treatment for wrinkles on his face. Beforegoing to sleep, the patient applies an electrically conductivemoisturizer to the area were wrinkles are visible. The patient then putson a mask that includes a multi-array matrix of biocompatiblemicrocells. The multi-array matrix produces a LLEC when it contacts themoisturizer. The patient repeats this each night. After 30 days oftreatment the patient's wrinkles are visibly reduced.

Example 10 Reduction of Facial Wrinkles in Combination with BOTOX®

A 44 year old male seeks treatment for “frown lines” between hiseyebrows. Following BOTOX® injection, the patient applies anelectrically conductive moisturizer to the area where the injectionswere administered. The patient then puts on a mask that includes amulti-array matrix of biocompatible microcells such that the matrixcontacts the moisturizer which contacts the skin. The multi-array matrixproduces a LLEC when it contacts the moisturizer. The patient repeatsthis each night. After 10 days of treatment the patient's wrinkles arevisibly reduced.

Example 11 Reduction of Facial Wrinkles in Combination with a DermalFiller

A 61 year old female seeks treatment for forehead wrinkles. Followingdermal filler injection, the patient applies an electrically conductivecream to the area where the injections were administered. The patientthen puts on a mask that includes a multi-array matrix of biocompatiblemicrocells such that the matrix contacts the moisturizer. Themulti-array matrix produces a LLEC when it contacts the cream. Thepatient repeats this each night. After 10 days of treatment thepatient's wrinkles are visibly reduced.

Example 12 Treatment of Acne

To treat acne of the back, the patient wears a three-layer vest thatcovers only the back as shown in FIG. 10. The vest consists of a layerof a multi-array matrix of biocompatible microcells as described hereinbacked by a hydration layer of polyvinyl alcohol fibers fabricated intoa uniform, spongelike open cell pore structure. An outer layer of awaterproof polyester fabric surrounds the hydration layer. A compressionshirt is worn over the vest to provide intimate contact between theelectrodes and the skin. The vest is soaked in warm water prior toplacement on the patient for the night. The material remains hydratedand produces voltage for over 24 hours (see FIGS. 11 and 12).

The material is cool to the touch but does not feel wet. A dry shirt canbe worn over the vest.

Example 13 Treatment of Acne

A fabric sheet is made with printed electrodes as described herein,using the pattern described in FIG. 1. The electrodes are printed on thefabric, then the fabric electrode sheet is attached to the inside of ashirt. The seam between the shirt and the bottom of the electrode sheetis left open. A layer of velcro is attached to the bottom of the shirtand the bottom of the electrode sheet so that the two can be sealedtogether. A layer of hydration material is inserted into the pocketbetween the shirt and the electrode sheet. The velcro seals thehydration sheet into the space between the shirt and the electrodesheet.

The hydration material is soaked for 15 minutes in distilled water inorder to hydrate the sheet. When the hydration is complete, thehydration sheet is placed back in the pocket between the shirt and theelectrodes and sealed in place using the velcro tabs. The patient thenputs on the shirt and goes to sleep for the night. The shirt is dry whenthe patient puts it on. The electrode surface will be damp and will feelcool to the touch. The patient sleeps with the shirt on. After 2 weeksof wearing the shirt for two hours each day, the appearance of acne isreduced.

Example 14 Treatment of Acne

A shirt is made with printed electrodes as described herein, using thepattern described in FIG. 1. The shirt is kept hydrated during the nightby a second looser fitting shirt that is worn over the top of the firstshirt. The second shirt has an inside layer of hydration material and anoutside layer of non-permeable fabric to hold the water inside. Bothshirts would be worn all night by the patient. After 2 weeks of nightlywear, the appearance of acne is reduced.

Example 15 Treatment of Acne

A shirt is made with printed electrodes as described herein, using thepattern described in FIG. 1. Prior to donning the shirt, the patientapplies a conductive anti-active agent to his skin in areas where acneis visible. The conductive anti-active agent is reapplied to thepatient's skin each night. After 2 weeks of nightly wear, the appearanceof acne is reduced.

Example 16 Treatment of Acne

A mask as seen in FIG. 9 is made with printed electrodes as describedherein, using the pattern described in FIG. 1. Prior to donning themask, the patient applies a conductive anti-active agent to the skinaround his cheeks in areas where acne is visible. The conductiveanti-active agent is reapplied to the patient's skin each night. After 2weeks of nightly wear, the appearance of acne is reduced.

Example 17 Modulation of Bacterial Gene Expression and Enzyme Activity

Treatment of biofilms presents a major challenge, because bacterialiving within them enjoy increased protection against host immuneresponses and are markedly more tolerant to antibiotics. Bacteriaresiding within biofilms are encapsulated in an extracellular matrix,consisting of several components including polysaccharides, proteins andDNA which acts as a diffusion barrier between embedded bacteria and theenvironment thus retarding penetration of antibacterial agents.Additionally, due to limited nutrient accessibility, thebiofilm-residing bacteria are in a physiological state of low metabolismand dormancy increasing their resistance towards antibiotic agents.

Chronic wounds present an increasing socio-economic problem and anestimated 1-2% of western population suffers from chronic ulcers andapproximately 2-4% of the national healthcare budget in developedcountries is spent on treatment and complications due to chronic wounds.The incidence of non-healing wounds is expected to rise as a naturalconsequence of longer lifespan and progressive changes in lifestyle likeobesity, diabetes, and cardiovascular disease. Non-healing skin ulcersare often infected by biofilms. Multiple bacterial species reside inchronic wounds; with Pseudomonas aeruginosa, especially in largerwounds, being the most common. P. aeruginosa is suspected to delayhealing of leg ulcers. Also, surgical success with split graft skintransplantation and overall healing rate of chronic venous ulcers ispresumably reduced when there is clinical infection by P. aeruginosa.

P. aeruginosa biofilm is often associated with chronic wound infection.The BED (“BED” or “bioelectric device” or PROCELLERA® as disclosedherein) consists of a matrix of silver-zinc coupled biocompatiblemicrocells, which in the presence of conductive wound exudate activatesto generate an electric field (0.3-0.9V). Growth (measured as O.D andcfu) of pathogenic Pseudomonas aeruginosa strain PAO1 in LB media wasmarkedly arrested in the presence of the BED (p<0.05, n=4). PAO1 biofilmwas developed in vitro using a polycarbonate filter model. Grownovernight in LB medium at 37° C. bacteria were cultured on sterilepolycarbonate membrane filters placed on LB agar plates and allowed toform a mature biofilm for 48 h. The biofilm was then exposed to BED orplacebo for the following 24 h. Structural characterization usingscanning electron microscopy demonstrated that the BED markedlydisrupted biofilm integrity as compared to no significant effectobserved using a commercial silver dressing commonly used for woundcare. Staining of extracellular polymeric substance, PAO1 staining, anda vital stain demonstrated a decrease in biofilm thickness and number oflive bacterial cells in the presence of BED (n=4). BED repressed theexpression of quorum sensing genes lasR and rhlR (p<0.05, n=3). BED wasalso found to generate micromolar amounts of superoxide (n=3), which areknown reductants and repress genes of the redox sensing multidrug effluxsystem mexAB and mexEF (n=3, p<0.05). BED also down-regulated theactivity of glycerol-3-phosphate dehydrogenase, an electric fieldsensitive enzyme responsible for bacterial respiration, glycolysis, andphospholipid biosynthesis (p<0.05, n=3).

Materials and Methods

In-Vitro Biofilm Model

PAO1 biofilm was developed in vitro using a polycarbonate filter model.Cells were grown overnight in LB medium at 37° C. bacteria were culturedon sterile polycarbonate membrane filters placed on LB agar plates andallowed to form a mature biofilm for 48 h. The biofilm was then exposedto BED or placebo for the following 24 h.

Energy Dispersive X-Ray Spectroscopy (EDS)

EDS elemental analysis of the Ag/ZN BED was performed in anenvironmental scanning electron microscope (ESEM, FEI XL-30) at 25 kV. Athin layer of carbon was evaporated onto the surface of the dressing toincrease the conductivity.

Scanning Electron Microscopy

Biofilm was grown on circular membranes and was then fixed in a 4%formaldehyde/2% glutaraldehyde solution for 48 hours at 4° C., washedwith phosphate-buffered saline solution buffer, dehydrated in a gradedethanol series, critical point dried, and mounted on an aluminum stub.The samples were then sputter coated with platinum (Pt) and imaged withthe SEM operating at 5 kV in the secondary electron mode (XL 30S; FEG,FEI Co., Hillsboro, Oreg.).

Live/Dead Staining

The LIVE/DEAD BacLight Bacterial Viability Kit for microscopy andquantitative assays was used to monitor the viability of bacterialpopulations. Cells with a compromised membrane that are considered to bedead or dying stain red, whereas cells with an intact membrane staingreen.

EPR Spectroscopy

EPR measurements were performed at room temperature using a Bruker ER300 EPR spectrometer operating at X-band with a TM 110 cavity. Themicrowave frequency was measured with an EIP Model 575 source-lockingmicrowave counter (EIP Microwave, Inc., San Jose, Calif.). Theinstrument settings used in the spin trapping experiments were asfollows: modulation amplitude, 0.32 G; time constant, 0.16 s; scan time,60 s; modulation frequency, 100 kHz; microwave power, 20 mW; microwavefrequency, 9.76 GHz. The samples were placed in a quartz EPR flat cell,and spectra were recorded at ambient temperature (25° C.). Serial 1-minEPR acquisitions were performed. The components of the spectra wereidentified, simulated, and quantitated as reported. The double integralsof DEPMPO experimental spectra were compared with those of a 1 mM TEMPOsample measured under identical settings to estimate the concentrationof superoxide adduct.

Quantification of mRNA and miRNA Expression

Total RNA, including the miRNA fraction, was isolated using Norgen RNAisolation kit, according to the manufacturers protocol. Gene expressionlevels were quantified with real-time PCR system and SYBR Green (AppliedBiosystems) and normalized to nadB and proC as housekeeping genes.Expression levels were quantified employing the 2 (−ΔΔct) relativequantification method.

Glycerol-3-Phosphate Dehydrogenase Assay

The glycerol-3-phosphate dehydrogenase assay was performed using anassay kit from Biovision, Inc. following manufacturer's instructions.Briefly, cells (˜1×10⁶) were homogenized with 200 μl ice cold GPDH Assaybuffer for 10 minutes on ice and the supernatant was used to measureO.D. and GPDH activity calculated from the results.

Statistics

Control and treated samples were compared by paired t test. Student's ttest was used for all other comparison of difference between means.P<0.05 was considered significant.

Ag/Zn BED Disrupts P. aeruginosa Biofilm

To validate the chemical composition of the dressing, we collected highresolution electron micrographs using an environmental scanning electronmicroscope. Our element maps indicate that silver particles areconcentrated in the golden dots of the polyester cloth, while zincparticles are concentrated in the grey dots.

As illustrated in FIG. 14A, P. aeruginosa was grown in round bottomtubes in LB medium with continuous shaking and absorbance was measuredby calculating optical density at 600 nm at different time points. Itwas observed that Ag/Zn BED and the control dressing with equal amountof silver inhibited bacterial growth (n=4) (FIG. 14B,C). When bacteriais grown in an agar plate with Ag/Zn BED dressing or placebo embedded inthe agar, the zone of inhibition is clearly visible in the case of Ag/ZnBED thus demonstrating its bacteriostatic property, while placebo withsilver dressing showed a smaller zone of inhibition, indicating theeffect role of electric field as compared to topical contact. (FIG.14D). However, as evident from scanning electron microscope images (FIG.15); EPS staining (FIG. 16); and live/dead staining (FIG. 17), Ag/Zn BEDdisrupts biofilm much better while silver does not have any effect onbiofilm disruption. Silver has been recognized for its antimicrobialproperties for centuries. Most studies on the antibacterial efficacy ofsilver, with particular emphasis on wound healing, have been performedon planktonic bacteria. Silver ions, bind to and react with proteins andenzymes, thereby causing structural changes in the bacterial cell walland membranes, leading to cellular disintegration and death of thebacterium. Silver also binds to bacterial DNA and RNA, therebyinhibiting the basal life processes.

Silver is effective against mature biofilms of P. aeruginosa, but onlyat a high silver concentration. A concentration of 5-10 μg/mL silversulfadiazine has been reported to eradicate biofilm whereas a lowerconcentration (1 μg/mL) had no effect. Therefore, the concentration ofsilver in currently available wound dressings is much too low fortreatment of chronic biofilm wounds. FIG. 18 shows PAO1 staining of thebiofilm demonstrating the lack of elevated mushroom like structures inthe Ag/Zn BED treated sample.

Ag/Zn BED Down-Regulates Quorum Sensing Genes

The pathogenicity of P. aeruginosa is attributable to an arsenal ofvirulence factors. The production of many of these extracellularvirulence factors occurs only when the bacterial cell density hasreached a threshold (quorum). Quorum sensing is controlled primarily bytwo cell-to-cell signaling systems, called las and rhl, which are bothcomposed of a transcriptional regulator (LasR and RhlR, respectively)and an autoinducer synthase (LasI and RhlI, respectively). In P.aeruginosa, LasI produces 3OC12-HSL. LasR, then, responds to this signaland the LasR:3OC12-HSL complex activates transcription of many genesincluding rhlR, which encodes a second quorum sensing receptor, RhlRwhich binds to autoinducer C4-HSL produced by RhlI. RhlR:C4-HSL alsodirects a large regulon of genes. P. aeruginosa defective in QS iscompromised in their ability to form biofilms. Quorum sensing inhibitorsincrease the susceptibility of the biofilms to multiple types ofantibiotics.

To test the effect of the electric field on quorum sensing genes, wesubjected the mature biofilm to the Ag/Zen BED or placebo for 12 hoursand looked at gene expression levels. We selected an earlier time point,because by 24 hours, as in earlier experiments, most bacteria underAg/Zn BED treatment were dead. We found a significant down regulation oflasR and rhlR (n=4, p<0.05). lasR transcription has been reported toweakly correlate with the transcription of lasA, lasB, toxA and aprA. Wedid not, however, find any significant difference in their expressionlevels at this time point, although we found them down regulated in theAg/Zn BED treated samples at the 24 hour time point (data not shown).(FIG. 19).

Ag/Zn BED Represses the Redox Sensing Multidrug Efflux System in P.aeruginosa

Ag/Zn BED acts as a reducing agent and reduces protein thiols. Oneelectron reduction of dioxygen O₂, results in the production ofsuperoxide anion. Molecular oxygen (dioxygen) contains two unpairedelectrons. The addition of a second electron fills one of its twodegenerate molecular orbitals, generating a charged ionic species withsingle unpaired electrons that exhibit paramagnetism. Superoxide anion,which can act as a biological reductant and can reduce disulfide bonds,is finally converted to hydrogen peroxide is known to have bactericidalproperties. Here, we used electron paramagnetic resonance (EPR) todetect superoxide directly upon exposure to the bioelectric dressing.Superoxide spin trap was carried out using DEPMPO(2-(diethoxyphosphoryl)-2-methyl-3,4-dihydro-2H-pyrrole 1-oxide) and ˜1μM superoxide anion production was detected upon 40 mins of exposure toAg/Zn BED (FIG. 20). MexR and MexT are two multidrug efflux regulatorsin P. aeruginosa which uses an oxidation-sensing mechanism. Oxidation ofboth MexR and MexT results in formation of intermolecular disulfidebonds, which activates them, leading to dissociation from promoter DNAand de-repression of MexAB-oprM and MexEF-oprN respectively, while in areduced state, they do not transcribe the operons. Induction of Mexoperons leads not only to increased antibiotic resistance but also torepression of the quorum sensing cascades and several virulence factors.We observe down-regulation of the downstream Mex genes MexA, MexB, MexEand MexF (but not MexC and MexD) (n=4, p<0.05), in Ag/Zn BED treatedsamples, inactive forms of MexR and MexT in their reduced states. Toconfirm the reducing activity of the Ag/Zn BED, the experiments wererepeated with 10 mM DTT and similar results were observed. (FIG. 21).

Ag/Zn BED Diminishes Glycerol-3-Phosphate Dehydrogenase Enzyme Activity

Electric fields can affect molecular charge distributions on manyenzymes. Glycerol-3-phosphate dehydrogenase is an enzyme involved inrespiration, glycolysis, and phospholipid biosynthesis and is expectedto be influenced by external electric fields in P. aeruginosa. Weobserved significantly diminished glycerol-3-phosphate dehydrogenaseenzyme activity by treating P. aeruginosa biofilm to the Ag/Zn BED for12 hours (n=3). (FIG. 22).

Example 18 LLEC Influence on Biofilm Properties

In this study ten clinical wound pathogens associated with chronic woundinfections were used for evaluating the anti-biofilm properties of aLLEC. Hydrogel and drip-flow reactor (DFR) biofilm models were employedfor the efficacy evaluation of the wound dressing in inhibitingbiofilms. Biofilms formed with Acinetobacter baumannii, Corynebacteriumamycolatum, Escherichia coli, Enterobacter aerogenes, Enterococcusfaecalis CI 4413, Klebsiella pneumonia, Pseudomonas aeruginosa, Serratiamarcescens, Staphylococcus aureus, and Streptococcus equi clinicalisolates were evaluated. For antimicrobial susceptibility testing ofbiofilms, 10⁵ CFU/mL bacteria was used in both biofilm models. Forpoloxamer hydrogel model, the LLECs (25 mm diameter) were applieddirectly onto the bacterial biofilm developed onto 30% poloxamerhydrogel and Muller-Hinton agar plates, and incubated at 37° C. for 24 hto observe any growth inhibition. In the DFR biofilm model, bacteriawere deposited onto polycarbonate membrane as abiotic surface, andsample dressings were applied onto the membrane. The DFR biofilm wasincubated in diluted trypticase soy broth (TSB) at room temperature for72 h. Biofilm formations were evaluated by crystal violet staining underlight microscopy, and anti-biofilm efficacy was demonstrated byreduction in bacterial numbers.

Example 19 Modulation of Mammalian Gene Expression and Enzyme Activity

Grown overnight in LB medium at 37° C., primary human dermal fibroblastsare cultured on sterile polycarbonate membrane filters placed on LB agarplates for 48 h. The cells are then exposed to BED or placebo for thefollowing 24 h. BED represses the expression of glyceraldehyde3-phosphate dehydrogenase. BED also down-regulates the activity ofglyceraldehyde 3-phosphate dehydrogenase.

Example 20 Modulation of Insect Gene Expression and Enzyme Activity

Grown overnight in LB medium at 37° C., drosophila S2 cells are culturedon sterile polycarbonate membrane filters placed on LB agar plates for48 h. The cells are then exposed to BED or placebo for the following 24h. BED represses the expression of insect P450 enzymes. BED alsodown-regulates the activity of insect P450 enzymes.

Example 21 Effect on Propionibacterium acnes

To determine the antimicrobial properties of the devices as disclosedherein, assessment of antibacterial finishes on textile materials wasconducted in accordance with the American Association of TextileChemists and Colorists (AATCC) Testing Methodology 100-1993. Themicrocurrent generating dressings were tested against multiple pathogensand successfully demonstrated antimicrobial properties against a broadspectrum of pathogens as shown in Table 1 below.

TABLE 1 In-vitro Percent Reduction in Microorganisms Microorganism %Reduction Escherichia coli 99.99 Aspergillus niger 99.27 Trichophytonmentagropytes 99.22 Vancomycin Resistant Enterococcus (VRE) 99.97Streptococcus pneumoniae 100 Methicillin Resistant Staph Aureus (MSRA)100 Staphylococcus aureus 100 Acinetobacter baumaanii 100 Trichophytonrubrum 99.99 Corynebacterium xerosis 100 Trichophyton ashii/inkin 99.97Pseudomonas aeruginosa 100 Candida albicans 99.98 Propionibacteriumacnes 100 Klebsiella Pneumonaie 100 Herpes Simplex Type 1 100 VaricellaVirus 99.98

Further studies will be performed as described:

Outcome Measures

Primary outcomes: Change in acne severity grade from baseline andsplit-back comparison as determined by Leeds Acne Grading System11 andblinded clinician extender evaluation. Change in acne severity based onmasked photographic assessment

Secondary outcomes: Patient subjective outcomes, per user assessmentsurvey. Patient assessment of wearability of study device

Study Design

This is a double-blinded, two-arm, same-patient, split-back, prospectivestudy investigating 50 patients presenting with acne vulgaris on theback; Patients will serve as their own controls and wear a vest (seeFIG. 13) as described herein comprising a substrate with biocompatibleelectrodes configured to generate an electric field, or in the presenceof a conductive solution, an electric current.

Study Site: This multicenter study will be conducted at two facilities;Paradise Valley Dermatology, Phoenix, Ariz. and Arizona AdvancedDermatology, Phoenix/Gilbert, Ariz.

Selection of Patients: The study population will include subjects age≥14 years ≤40 years.

Number of Patients: 50 subjects completing up to at least week 6 oftreatment will be considered evaluable. Subject population will includemale or female subjects of all ethnic groups. Parental consent fortreatment of minors will be obtained for subjects <18 years.

Inclusion Criteria:

-   -   a. Clinical diagnosis of mild or moderate acne vulgaris of the        back (Leeds scale grades 1-5)    -   b. Subjects age ≥14 years ≤40 years    -   c. Participants willing to undergo treatment and participate in        follow up evaluations. Participants willing to comply with study        procedures and willing to refrain from “picking” at lesions.    -   d. Participants with cell phones and willing to receive daily        text reminders to wear study device.    -   e. Participants falling outside the washout periods for topical        and systemic treatments and office procedures.    -   f. 4 weeks for topical agents on the back (corticosteroids,        retinoids and other acne treatments);    -   g. 8 weeks for office-based acne procedures on the back (for        chemical peeling, laser and light-based therapies).    -   h. 12 weeks for systemic drugs (corticosteroids and other acne        treatments)    -   i. Subjects requiring topical agents for the face or requiring        office-based acne procedures for the face will be included in        the study.    -   j. Participant must be able to read and understand informed        consent, and signs the informed consent

Exclusion Criteria

-   -   a. Clinical diagnosis of severe acne vulgaris of the back        requiring systemic drugs for management, as defined by Leeds        Scale Grades ≥6    -   b. Less than 14 years of age or over 40 years of age.    -   c. Patients receiving any topical or office-based acne        procedures within the washout period prior to study.    -   d. Participation in another clinical trial that involved the use        of an investigational drug or device that in the opinion of the        investigator would confound the results of this trial    -   e. Individuals with silver or zinc sensitivity    -   f. Active cancer    -   g. Immunosuppressive treatment    -   h. Clinically diagnosed hyperandrogenic state    -   i. Evidence of severe androgen excess (i.e., testosterone        levels >150 ng/dL)    -   j. Excessive back hair in male patients    -   k. Pregnant or nursing individuals    -   l. Patients with pacemakers    -   m. Cystic or nodular acne lesions    -   n. Use of medicated shampoos, body washes, exfoliants and benzyl        peroxide washes for 4 weeks prior to study start.    -   o. Any other medical condition in the clinician's opinion that        excludes the patient.    -   p. Geographical concerns (residence not within reasonable travel        distance) that would hamper compliance with required study        visits.    -   q. The Investigator believes that the subject will be unwilling        or unable to comply with study protocol requirements, including        application of study device and all study-related follow up        visit requirements.

The participants must answer “yes” on all inclusion criteria and mustanswer “no” on all exclusion criteria

13.6 Patient Recruitment

Patients treated at Paradise Valley Dermatology, Arizona AdvancedDermatology, Center for Dermatology, and the Skin and Cancer Center ofAZ presented with acne vulgaris of the back will be given theopportunity to enroll into the study. At time of screening, thephysician extender will document non-identifiers including date, sex,age, presence of back acne, and interest in participation in a study. Ifthere is an interest, the patients will be given a detailed explanationof the risks and benefits of the study.

Method of Obtaining Informed Consent

Written informed consent will be obtained from the patients by thephysicians, residents and/or staff present in the clinic who aretreating the individual patients

Plan of Study

The system to be used in the present study is a 2-part vest designed tocover the scapular area of the back (see FIG. 13). The outer vest iscomprised of vinyl, polyester trim and elastic materials and adjustablestraps. The vest contains a detachable absorbent pad comprised ofpolyvinyl acetate (PVA) lined with the multi-array matrix ofbiocompatible microcells printed on one half of the vest, and a placebopattern on the other half of the vest

Description of Study Procedures

Randomization

Patients will be randomized into Group X or Group Y. 25 patients will berandomized to receive vests with the multi-array matrix of biocompatiblemicrocells printed on the right half and placebo pattern on the lefthalf (Group X) and 25 patients will receive vests with multi-arraymatrix of biocompatible microcells printed on the left half and placeboon the right half (Group Y). The patient, investigators and physicianextenders (nurses, PA's, etc) will be blinded to the assigned side ofthe vest. Just prior to dissemination of study materials, theinvestigator will be provided with the randomization of treatment theparticipant will receive in the duration of the study, Group X or GroupY. A randomization key will be held at a secure location with the studysponsor. After final study analysis, the key will be revealed. In theevent the randomization is made apparent, a note will be documented inthe patient file. Patient will still be included in the study and studydata will still be considered evaluable.

Group Treatment Vest Configuration

Group X Multi-array matrix of biocompatible microcells on RIGHT side ofvest and placebo pattern on LEFT side of vest

Group Y Multi-array matrix of biocompatible microcells on LEFT side ofvest and placebo pattern on RIGHT side of vest

Study Materials

Participants will be provided the following study supplies:

-   -   a. Plastic vest replacements    -   b. Removable multi-array matrix of biocompatible microcells vest        pad replacements    -   c. Supply of compression shirts    -   d. Study Log    -   e. Carrying tote for materials

Participants will be instructed in the use of the multi-array matrix ofbiocompatible microcells vest, and will be supplied with detailedinstructions for use.

Device Application Method

Before going to bed, patient will be instructed to apply the vestembodiment according to the provided instructions. Patients will moistenthe pad of the vest and secure the vest to the body. They will wear aspandex t-shirt covering the entire vest. They will continue to wear thevest throughout the course of the night and remove the nighttime vest inthe morning after awakening.

Patient Compliance Monitoring

Patients will be reminded to wear their study device by documenting wearthrough a paper log and/or electronic technology platform.

Automated Patient Communications

An automated patient communication portal, i.e. Constant Contact,SolutionReach, PracticeMojo, etc. will be used for patient compliancepurposes. During the consent process, will have agreed to participate inautomated text reminders. To remind patients to wear the study deviceeach day, patients will receive a daily evening reminder on theircellular phone to wear the study device, as well as periodic remindersthroughout the week (˜3 times week) to change the vest pads. In the2-way communication platform, participants will be requested to text “C”for confirming compliance. To ensure patient confidentiality andprivacy, all text communications will comply with all applicable rulesand regulations—see Appendix 1.

Deviations:

Participants will be asked to document all deviations from studyprocedures, and log dates where the vest was not worn due to extenuatingcircumstances.

Study Period

-   -   a. Visit 0—Baseline Visit 1—Week 2    -   b. (Day 14±2), Visit 2—Week 4 (Day 28±2), Visit 3—Week 6    -   c. (Day 42±2).

Study Enrollment

Consent form/Minor Consent

Medical History

Baseline Acne Grading

Digital Photos

Skin Evaluation

Vest distribution

Standard Follow-up Procedures:

Digital Photos

Skin Evaluation

Acne Grading

Vest distribution

Standard Follow-up Procedures

Digital Photos

Skin Evaluation

Acne Grading

Vest distribution

Standard Follow-up Procedures

Digital Photos

Skin Evaluation

Acne Grading

End of Study

Clinic Visits

-   -   a. Patients will be seen at the clinic for a total of four        visits during the study, with baseline visit 0 serving as the        initial visit. Follow-up visits will occur when the participants        return to the clinic at weeks 2, 4 and 6 after the initial        baseline visit for their follow-up evaluations.

Clinic Assessments

Initial Intake

Basic patient demographic information will be collected at the initialstudy visit, including:

-   -   a. Age    -   b. Gender    -   c. Past acne treatments    -   d. Other skin conditions and/or relevant medical conditions

The following study procedures will be performed at each visit:

Clinician Acne Grading: At the initial and each follow-up visit, theacne lesions will assessed by a physician extender, who will be blindedto the patients' randomization. Five separate evaluations will beperformed at each visit and will be documented on case report form.

-   -   a. 1) Leeds Rating: Grading of acne severity will be conducted        according to the Leeds Acne Grading System, an overall        assessment of acne severity for use in routine clinic, which        grades patients on a scale of 0 (no acne) to 10 (the most        severe). For the purpose of this study, only patients falling in        between grades 1-5 will be included.

2) Clinical Evaluation

-   -   a. A standard skin assessment, evaluating presence of infection,        erythema, and irritation will be documented.

3) Lesion Count

-   -   a. The area of the back above the scapula will be divided into        four equal quadrants: Left upper, left lower, right upper and        right lower. The physician extender will count and document the        number of lesions observed in each quadrant.

4) Global assessment

-   -   a. Efficacy of each treatment will be investigated by global        assessment; the physician extender will assess if the global        acne appearance on each half of the back has improved, is the        same, or has worsened compared to the baseline photo.

5) Side by Side assessment

-   -   a. The physician extender will perform a “side-by-side”        evaluation to evaluate the appearance of acne of the left side        compared to the right side of the back, and document if the left        or right side of the back looks better, or the same.

Photograph

-   -   a. Standardized digital photography to capture progressive        changes in acne lesions over time will be performed at each        visit. A standard photography station will be set up at each        clinic site, with digital camera mounted on tripod. The back        will be photographed according to the following standards:    -   b. Angle: The photo is to be taken perpendicular to the back    -   c. Lighting: The photo is to be taken under the same lighting        conditions.    -   d. Height: The photo is to be taken at a standard height for        each patient, with height adjustable as needed.    -   e. Necessary information to accompany each photo: Label with        participant # and date    -   f. Number of photos: Photos will be obtained at each follow-up        visit; a total of 4 photographs will be taken: Baseline, Week 2,        Week 4, and Week 6 of study.    -   g. Pre and post-treatment photos will be overlaid for        comparison.

Patient Acne Assessment

-   -   a. At the follow-up visits, patients will evaluate photos of        their back at the current study visit and self-report on the        following: general appearance of acne, redness and discomfort of        acne lesions.    -   b. Patient clinical assessment will be documented on case report        form “Patient Acne Assessment” XV-060CRF-06.

Wearability Assessment

-   -   a. Participants will also answer a questionnaire at their last        visit to assess their experience with the vest. Participants        will rate comfort level (vest, strap, moisture, temperature,        sleep quality) and other observations during the study. Outcomes        will be captured on “Wearability assessment” XV-060CRF-08.

Blinded Clinician Photo Assessment

-   -   a. Photos will be graded per by two independent clinicians, with        a third clinician brought in if tie-breaking is required.        Clinicians will be provided digital photos of each participant,        with photos out of chronologic order

In the first series of four photos, clinicians will perform a “side byside” comparison evaluating left side of back versus right side of backin each of the photos, and selecting which side appears better,Left-Right- or if they appear the Same.

In the second series of three photos, clinicians will perform a globalassessment evaluating left half of back versus baseline photo, and righthalf of back versus baseline photo. Baseline photo is labeled butsubsequent photos are not in chronologic order.

Study Endpoint

The study will end 6 weeks after first day of enrollment in the study.All patients will be monitored for adverse side effects including butnot limited to infection, skin sensitivity, and worsening condition ofthe acne condition during the course of the study.

Study Outcomes: All patients will be assessed for:

-   -   a. Acne improvement via Leeds System and clinical skin        assessment    -   b. Visual acne improvement via digital photography    -   c. Wearability assessment via patient survey    -   d. Patient self assessment of acne clinical condition

In closing, it is to be understood that although aspects of the presentspecification are highlighted by referring to specific embodiments, oneskilled in the art will readily appreciate that these disclosedembodiments are only illustrative of the principles of the subjectmatter disclosed herein. Therefore, it should be understood that thedisclosed subject matter is in no way limited to a particularmethodology, protocol, and/or reagent, etc., described herein. As such,various modifications or changes to or alternative configurations of thedisclosed subject matter can be made in accordance with the teachingsherein without departing from the spirit of the present specification.Lastly, the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present disclosure, which is defined solely by the claims.Accordingly, embodiments of the present disclosure are not limited tothose precisely as shown and described.

Certain embodiments are described herein, including the best mode knownto the inventor for carrying out the methods and devices describedherein. Of course, variations on these described embodiments will becomeapparent to those of ordinary skill in the art upon reading theforegoing description. Accordingly, this disclosure includes allmodifications and equivalents of the subject matter recited in theclaims appended hereto as permitted by applicable law. Moreover, anycombination of the above-described embodiments in all possiblevariations thereof is encompassed by the disclosure unless otherwiseindicated herein or otherwise clearly contradicted by context.

Groupings of alternative embodiments, elements, or steps of the presentdisclosure are not to be construed as limitations. Each group member maybe referred to and claimed individually or in any combination with othergroup members disclosed herein. It is anticipated that one or moremembers of a group may be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion ordeletion occurs, the specification is deemed to contain the group asmodified thus fulfilling the written description of all Markush groupsused in the appended claims.

Unless otherwise indicated, all numbers expressing a characteristic,item, quantity, parameter, property, term, and so forth used in thepresent specification and claims are to be understood as being modifiedin all instances by the term “about.” As used herein, the term “about”means that the characteristic, item, quantity, parameter, property, orterm so qualified encompasses a range of plus or minus ten percent aboveand below the value of the stated characteristic, item, quantity,parameter, property, or term. Accordingly, unless indicated to thecontrary, the numerical parameters set forth in the specification andattached claims are approximations that may vary. At the very least, andnot as an attempt to limit the application of the doctrine ofequivalents to the scope of the claims, each numerical indication shouldat least be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Notwithstandingthat the numerical ranges and values setting forth the broad scope ofthe disclosure are approximations, the numerical ranges and values setforth in the specific examples are reported as precisely as possible.Any numerical range or value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements. Recitation of numerical ranges ofvalues herein is merely intended to serve as a shorthand method ofreferring individually to each separate numerical value falling withinthe range. Unless otherwise indicated herein, each individual value of anumerical range is incorporated into the present specification as if itwere individually recited herein.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the disclosure (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.,“such as”) provided herein is intended merely to better illuminate thedisclosure and does not pose a limitation on the scope otherwiseclaimed. No language in the present specification should be construed asindicating any non-claimed element essential to the practice ofembodiments disclosed herein.

Specific embodiments disclosed herein may be further limited in theclaims using consisting of or consisting essentially of language. Whenused in the claims, whether as filed or added per amendment, thetransition term “consisting of” excludes any element, step, oringredient not specified in the claims. The transition term “consistingessentially of” limits the scope of a claim to the specified materialsor steps and those that do not materially affect the basic and novelcharacteristic(s). Embodiments of the present disclosure so claimed areinherently or expressly described and enabled herein.

The invention claimed is:
 1. A device for treating acne comprising alaminate comprising an elastic outer layer and an in-elastic innerlayer, said inner layer comprising a substrate comprising adiscontinuous region, wherein said discontinuous region is perpendicularto a long axis of the device and does not extend to a perimeter of thedevice, said substrate comprising one or more biocompatible electrodesconfigured to generate at least one of a low level electric field (LLEF)or low level electric current (LLEC).
 2. The device of claim 1 whereinthe biocompatible electrodes comprise a first array comprising a patternof microcells formed from a first conductive material, and a secondarray comprising a pattern of microcells formed from a second conductivematerial.
 3. The device of claim 2 wherein the first conductive materialand the second conductive material comprise the same material.
 4. Thedevice of claim 3 wherein the first and second array each comprise adiscrete circuit.
 5. The device of claim 4, further comprising a powersource.
 6. The device of claim 2 wherein the first array and the secondarray spontaneously generate a LLEF.
 7. The device of claim 6, furthercomprising an electrolytic solution, wherein the first array and thesecond array spontaneously generate a LLEC when contacted with saidelectrolytic solution.
 8. The device of claim 2, wherein said firstconductive material comprises silver.
 9. A method for treating acnecomprising affixing the device of claim 1 to an area where acnetreatment is desired.
 10. A device for rejuvenating skin comprising alaminate comprising an elastic outer layer and an in-elastic innerlayer, said inner layer comprising a substrate comprising adiscontinuous region, wherein said discontinuous region is perpendicularto a long axis of the device and does not extend to a perimeter of thedevice, said substrate comprising biocompatible electrodes capable ofgenerating at least one of a low level electric field (LLEF) or lowlevel micro current (LLEC); and a conductive fluid in contact with theelectrodes.
 11. The device of claim 10 wherein the biocompatibleelectrodes comprise a first array comprising a pattern of microcellsformed from a first conductive material, and a second array comprising apattern of microcells formed from a second conductive material.
 12. Thedevice of claim 11 wherein the first conductive material and the secondconductive material comprise the same material.
 13. The device of claim12 wherein the first and second array each comprise a discrete circuit.14. The device of claim 13, further comprising a power source.
 15. Thedevice of claim 11, wherein said first conductive material comprisessilver.